U.S. patent application number 10/215328 was filed with the patent office on 2003-09-25 for multigene expression vectors for the biosynthesis of products via multienzyme biological pathways.
This patent application is currently assigned to Monsanto Technology LLC. Invention is credited to Hao, Ming, Houmiel, Kathryn L., Mitsky, Timothy A., Reiser, Steven E., Slater, Steven C..
Application Number | 20030182678 10/215328 |
Document ID | / |
Family ID | 22406237 |
Filed Date | 2003-09-25 |
United States Patent
Application |
20030182678 |
Kind Code |
A1 |
Mitsky, Timothy A. ; et
al. |
September 25, 2003 |
Multigene expression vectors for the biosynthesis of products via
multienzyme biological pathways
Abstract
The use of multigene vectors for the preparation of transformed
host cells and plants is disclosed. Multigene vectors reduce the
number of transformations required, and leads to increased
production of polyhydroxyalkanoate polymer in the resulting
transformed host cells and plants.
Inventors: |
Mitsky, Timothy A.;
(Maryland Heights, MO) ; Slater, Steven C.;
(Acton, MA) ; Reiser, Steven E.; (St. Louis,
MO) ; Hao, Ming; (St. Louis, MO) ; Houmiel,
Kathryn L.; (Chesterfield, MO) |
Correspondence
Address: |
Patricia A. Kammerer, Esq.
HOWREY SIMON ARNOLD & WHITE, LLP
750 Bering Drive
Houston
TX
77057-2198
US
|
Assignee: |
Monsanto Technology LLC
|
Family ID: |
22406237 |
Appl. No.: |
10/215328 |
Filed: |
August 8, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10215328 |
Aug 8, 2002 |
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09517978 |
Mar 3, 2000 |
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6448473 |
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60123015 |
Mar 5, 1999 |
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Current U.S.
Class: |
800/278 ;
435/135; 435/320.1; 435/419 |
Current CPC
Class: |
C12N 15/8216 20130101;
C12N 15/8243 20130101 |
Class at
Publication: |
800/278 ;
435/320.1; 435/135; 435/419 |
International
Class: |
A01H 001/00; C12P
007/62; C12N 015/87; C12N 005/04 |
Claims
What is claimed is:
1. An isolated nucleic acid segment comprising: a first nucleic
acid sequence encoding a polyhydroxyalkanoate synthase protein; a
second nucleic acid sequence encoding a .beta.-ketoacyl reductase
protein; and a third nucleic acid sequence encoding a
.beta.-ketothiolase protein.
2. The isolated nucleic acid segment of claim 1, further comprising
a fourth nucleic acid sequence encoding a threonine deaminase
protein.
3. The isolated nucleic acid segment of claim 1, further comprising
a fourth nucleic acid sequence encoding a deregulated threonine
deaminase protein.
4. The isolated nucleic acid segment of claim 1, wherein: the first
nucleic acid sequence further encodes a chloroplast transit
peptide; the second nucleic acid sequence further encodes a
chloroplast transit peptide; and the third nucleic acid sequence
further encodes a chloroplast transit peptide.
5. A recombinant vector comprising operatively linked in the 5' to
3' direction: a promoter that directs transcription of a first
nucleic acid sequence, a second nucleic acid sequence, and a third
nucleic acid sequence; a first nucleic acid sequence; a second
nucleic acid sequence; a third nucleic acid sequence; a 3'
transcription terminator; and a 3' polyadenylation signal sequence;
wherein: the first nucleic acid sequence, second nucleic acid
sequence, and third nucleic acid sequence encode different
proteins; and the first nucleic acid sequence, second nucleic acid
sequence, and third nucleic acid sequence are independently
selected from the group consisting of a nucleic acid sequence
encoding a polyhydroxyalkanoate synthase protein, a nucleic acid
sequence encoding a .beta.-ketoacyl reductase protein, and a
nucleic acid sequence encoding a .beta.-ketothiolase protein.
6. The recombinant vector of claim 5, wherein the promoter directs
transcription of the first nucleic acid sequence, the second
nucleic acid sequence, and the third nucleic acid sequence in
plants.
7. The recombinant vector of claim 5, wherein the promoter is a
viral promoter.
8. The recombinant vector of claim 5, wherein the promoter is a CMV
35S promoter, an enhanced CMV 35S promoter, or an FMV 35S
promoter.
9. The recombinant vector of claim 5, wherein the promoter is an
enhanced CMV 35S promoter.
10. The recombinant vector of claim 5, wherein the promoter is a
tissue specific promoter.
11. The recombinant vector of claim 5, wherein the promoter is a
Lesquerella hydroxylase promoter or a 7S conglycinin promoter.
12. The recombinant vector of claim 5, wherein the promoter is a
Lesquerella hydroxylase promoter.
13. The recombinant vector of claim 5, wherein: the first nucleic
acid sequence further encodes a chloroplast transit peptide; the
second nucleic acid sequence further encodes a chloroplast transit
peptide; and the third nucleic acid sequence further encodes a
chloroplast transit peptide.
14. A recombinant vector comprising: a first element comprising
operatively linked in the 5' to 3' direction: a first promoter that
directs transcription of a first nucleic acid sequence; a first
nucleic acid sequence encoding a polyhydroxyalkanoate synthase
protein; a first 3' transcription terminator; and a first 3'
polyadenylation signal sequence; a second element comprising
operatively linked in the 5' to 3' direction: a second promoter
that directs transcription of a second nucleic acid sequence; a
second nucleic acid sequence encoding a .beta.-ketoacyl reductase
protein; a second 3' transcription terminator; and a second 3'
polyadenylation signal sequence; and a third element comprising
operatively linked in the 5' to 3' direction: a third promoter that
directs transcription of a third nucleic acid sequence; a third
nucleic acid sequence encoding a .beta.-ketothiolase protein; a
third 3' transcription terminator; and a third 3' polyadenylation
signal sequence.
15. The recombinant vector of claim 14, wherein the
.beta.-ketothiolase protein: catalyzes the condensation of two
molecules of acetyl-CoA to produce acetoacetyl-CoA; and catalyzes
the condensation of acetyl-CoA and propionyl-CoA to produce
.beta.-ketovaleryl-CoA.
16. The recombinant vector of claim 14, wherein the .beta.-ketoacyl
reductase protein: catalyzes the reduction of acetoacetyl-CoA to
.beta.-hydroxybutyryl-CoA; and catalyzes the reduction of
.beta.-ketovaleryl-CoA to .beta.-hydroxyvaleryl-CoA.
17. The recombinant vector of claim 14, wherein the
polyhydroxyalkanoate synthase protein is selected from the group
consisting of: a polyhydroxyalkanoate synthase protein that
catalyzes the incorporation of .beta.-hydroxybutyryl-CoA into
P(3HB) polymer; and a polyhydroxyalkanoate synthase protein that
catalyzes the incorporation of .beta.-hydroxybutyryl-CoA and
.beta.-hydroxyvaleryl-CoA into P(3HB-co-3HV) copolymer.
18. The recombinant vector of claim 14, wherein: the
.beta.-ketothiolase protein comprises a transit peptide sequence
that directs transport of the .beta.-ketothiolase protein to the
plastid; the .beta.-ketoacyl reductase protein comprises a transit
peptide sequence that directs transport of the .beta.-ketoacyl
reductase protein to the plastid; and the polyhydroxyalkanoate
synthase protein comprises a transit peptide sequence that directs
transport of the polyhydroxyalkanoate synthase protein to the
plastid.
19. The recombinant vector of claim 14, further comprising a
nucleic acid sequence encoding a threonine deaminase protein.
20. The recombinant vector of claim 14, further comprising a
nucleic acid sequence encoding a deregulated threonine deaminase
protein.
21. The recombinant vector of claim 14, wherein: the first promoter
directs transcription of the first nucleic acid sequence in plants;
the second promoter directs transcription of the second nucleic
acid sequence in plants; and the third promoter directs
transcription of the third nucleic acid sequence in plants.
22. The recombinant vector of claim 14, wherein the first promoter,
second promoter, and third promoter are viral promoters.
23. The recombinant vector of claim 14, wherein: the first promoter
is a CMV 35S promoter, an enhanced CMV 35S promoter, or an FMV 35S
promoter; the second promoter is a CMV 35S promoter, an enhanced
CMV 35S promoter, or an FMV 35S promoter; and the third promoter is
a CMV 35S promoter, an enhanced CMV 35S promoter, or an FMV 35S
promoter.
24. The recombinant vector of claim 14, wherein: the first promoter
is an enhanced CMV 35S promoter; the second promoter is an enhanced
CMV 35S promoter; and the third promoter is an enhanced CMV 35S
promoter.
25. The recombinant vector of claim 14, wherein: the first promoter
is a tissue specific promoter; the second promoter is a tissue
specific promoter; and the third promoter is a tissue specific
promoter.
26. The recombinant vector of claim 14, wherein: the first promoter
is a Lesquerella hydroxylase promoter or a 7S conglycinin promoter;
the second promoter is a Lesquerella hydroxylase promoter or a 7S
conglycinin promoter; and the third promoter is a Lesquerella
hydroxylase promoter or a 7S conglycinin promoter.
27. The recombinant vector of claim 14, wherein: the first promoter
is a Lesquerella hydroxylase promoter; the second promoter is a
Lesquerella hydroxylase promoter; and the third promoter is a
Lesquerella hydroxylase promoter.
28. The recombinant vector of claim 14, wherein: the first nucleic
acid sequence further encodes a chloroplast transit peptide; the
second nucleic acid sequence further encodes a chloroplast transit
peptide; and the third nucleic acid sequence further encodes a
chloroplast transit peptide.
29. A transformed host cell comprising a recombinant vector,
wherein the recombinant vector comprises: a first element
comprising operatively linked in the 5' to 3' direction: a first
promoter that directs transcription of a first nucleic acid
sequence; a first nucleic acid sequence encoding a
polyhydroxyalkanoate synthase protein; a first 3' transcription
terminator; and a first 3' polyadenylation signal sequence; a
second element comprising operatively linked in the 5' to 3'
direction: a second promoter that directs transcription of a second
nucleic acid sequence; a second nucleic acid sequence encoding a
.beta.-ketoacyl reductase protein; a second 3' transcription
terminator; and a second 3' polyadenylation signal sequence; and a
third element comprising operatively linked in the 5' to 3'
direction: a third promoter that directs transcription of a third
nucleic acid sequence; a third nucleic acid sequence encoding a
.beta.-ketothiolase protein; a third 3' transcription terminator;
and a third 3' polyadenylation signal sequence.
30. The transformed host cell of claim 29, wherein the transformed
host cell is a bacterial cell.
31. The transformed host cell of claim 29, wherein the transformed
host cell is a fungal cell.
32. The transformed host cell of claim 29, wherein the transformed
host cell is a plant cell.
33. The transformed host cell of claim 29, wherein: the first
nucleic acid sequence further encodes a chloroplast transit
peptide; the second nucleic acid sequence further encodes a
chloroplast transit peptide; and the third nucleic acid sequence
further encodes a chloroplast transit peptide.
34. A transformed host cell comprising: a first element comprising
operatively linked in the 5' to 3' direction: a first promoter that
directs transcription of a first nucleic acid sequence; a first
nucleic acid sequence encoding a polyhydroxyalkanoate synthase
protein; a first 3' transcription terminator; and a first 3'
polyadenylation signal sequence; a second element comprising
operatively linked in the 5' to 3' direction: a second promoter
that directs transcription of a second nucleic acid sequence; a
second nucleic acid sequence encoding a .beta.-ketoacyl reductase
protein; a second 3' transcription terminator; and a second 3'
polyadenylation signal sequence; and a third element comprising
operatively linked in the 5' to 3' direction: a third promoter that
directs transcription of a third nucleic acid sequence; a third
nucleic acid sequence encoding a .beta.-ketothiolase protein; a
third 3' transcription terminator; and a third 3' polyadenylation
signal sequence; wherein the first element, second element, and
third element are cointegrated between a single left Ti border
sequence and a single right Ti border sequence.
35. The transformed host cell of claim 34, wherein the transformed
host cell is a fungal cell.
36. The transformed host cell of claim 34, wherein the transformed
host cell is a plant cell.
37. The transformed host cell of claim 34, wherein the transformed
host cell is a tobacco, wheat, potato, Arabidopsis, corn, soybean,
canola, oil seed rape, sunflower, flax, peanut, sugarcane,
switchgrass, or alfalfa cell.
38. The transformed host cell of claim 34, wherein: the first
nucleic acid sequence further encodes a chloroplast transit
peptide; the second nucleic acid sequence further encodes a
chloroplast transit peptide; and the third nucleic acid sequence
further encodes a chloroplast transit peptide.
39. A transformed plant comprising: a first element comprising
operatively linked in the 5' to 3' direction: a first promoter that
directs transcription of a first nucleic acid sequence; a first
nucleic acid sequence encoding a polyhydroxyalkanoate synthase
protein; a first 3' transcription terminator; and a first 3'
polyadenylation signal sequence; a second element comprising
operatively linked in the 5' to 3' direction: a second promoter
that directs transcription of a second nucleic acid sequence; a
second nucleic acid sequence encoding a .beta.-ketoacyl reductase
protein; a second 3' transcription terminator; and a second 3'
polyadenylation signal sequence; and a third element comprising
operatively linked in the 5' to 3' direction: a third promoter that
directs transcription of a third nucleic acid sequence; a third
nucleic acid sequence encoding a .beta.-ketothiolase protein; a
third 3' transcription terminator; and a third 3' polyadenylation
signal sequence; wherein the first element, second element, and
third element are cointegrated between a single left Ti border
sequence and a single right Ti border sequence.
40. The transformed plant of claim 39, wherein the transformed
plant is a tobacco, wheat, potato, Arabidopsis, corn, soybean,
canola, oil seed rape, sunflower, flax, peanut, sugarcane,
switchgrass, or alfalfa plant.
41. The transformed plant of claim 39, wherein: the first nucleic
acid sequence further encodes a chloroplast transit peptide; the
second nucleic acid sequence further encodes a chloroplast transit
peptide; and the third nucleic acid sequence further encodes a
chloroplast transit peptide.
42. A method of preparing transformed host cells, the method
comprising: selecting a host cell; transforming the selected host
cell with a recombinant vector comprising: a first element
comprising operatively linked in the 5' to 3' direction: a first
promoter that directs transcription of the first nucleic acid
sequence; a first nucleic acid sequence encoding a
polyhydroxyalkanoate synthase protein; a first 3' transcription
terminator; and a first 3' polyadenylation signal sequence; a
second element comprising operatively linked in the 5' to 3'
direction: a second promoter that directs transcription of the
second nucleic acid sequence; a second nucleic acid sequence
encoding a .beta.-ketoacyl reductase protein; a second 3'
transcription terminator; and a second 3' polyadenylation signal
sequence; and a third element comprising operatively linked in the
5' to 3' direction: a third promoter that directs transcription of
the third nucleic acid sequence; a third nucleic acid sequence
encoding a .beta.-ketothiolase protein; a third 3' transcription
terminator; and a third 3' polyadenylation signal sequence; and
obtaining transformed host cells; wherein the transformed host
cells produce polyhydroxyalkanoate polymer.
43. A method of preparing transformed host cells, the method
comprising: selecting a host cell; transforming the selected host
cell with a recombinant vector comprising operatively linked in the
5' to 3' direction: a promoter that directs transcription of a
first nucleic acid sequence, second nucleic acid sequence, and
third nucleic acid sequence; a first nucleic acid sequence; a
second nucleic acid sequence; a third nucleic acid sequence; a 3'
transcription terminator; and a 3' polyadenylation signal sequence;
and obtaining transformed host cells; wherein: the first nucleic
acid sequence, second nucleic acid sequence, and third nucleic acid
sequence encode different proteins; the first nucleic acid
sequence, second nucleic acid sequence, and third nucleic acid
sequence are independently selected from the group consisting of a
nucleic acid sequence encoding a polyhydroxyalkanoate synthase
protein, a nucleic acid sequence regenerating the transformed host
plant cells to produce transformed plants; wherein: the first
nucleic acid sequence, second nucleic acid sequence, and third
nucleic acid sequence encode different proteins; the first nucleic
acid sequence, second nucleic acid sequence, and third nucleic acid
sequence are independently selected from the group consisting of a
nucleic acid sequence encoding a polyhydroxyalkanoate synthase
protein, a nucleic acid sequence encoding a .beta.-ketoacyl
reductase protein, and a nucleic acid sequence encoding a
.beta.-ketothiolase protein; and the transformed plants produce
polyhydroxyalkanoate polymer.
46. A method of producing polyhydroxyalkanoate comprising:
obtaining the transformed host cell of claim 29 or claim 34;
culturing the transformed host cell under conditions suitable for
the production of polyhydroxyalkanoate; and recovering
polyhydroxyalkanoate from the transformed host cell.
47. The method of claim 46, wherein the polyhydroxyalkanoate is
poly(3-hydroxybutyrate), poly(4-hydroxybutyrate), or
poly(3-hydroxybutyrate-co-4-hydroxybutyrate).
48. A method of producing polyhydroxyalkanoate comprising:
obtaining the transformed plant of claim 39; growing the
transformed plant under conditions suitable for the production of
polyhydroxyalkanoate; and recovering polyhydroxyalkanoate from the
transformed plant.
49. The method of claim 48, wherein the polyhydroxyalkanoate is
poly(3-hydroxybutyrate), poly(3-hydroxyvalerate), or
poly(3-hydroxybutyrate-co-3-hydroxyvalerate).
Description
[0001] This application is based on U.S. Provisional Application
No. 60/123,015, filed Mar. 5, 1999.
FIELD OF THE INVENTION
[0002] The invention relates to the construction and use of
multigene expression vectors useful to enhance production of
materials by multienzyme pathways. In particular, the construction
and use of multigene vectors encoding proteins in the
polyhydroxyalkanoate biosynthetic pathway is disclosed.
BACKGROUND OF THE INVENTION
[0003] Metabolic engineering is a process by which the normal
metabolism of an organism is altered to change the concentration of
normal metabolites, or to create novel metabolites. This process
often involves introduction or alteration of numerous enzymatic
steps, and thus often requires introduction of multiple genes. An
efficient system for introducing and expressing multiple genes is
therefore desirable. In prokaryotes such as Escherichia coli,
introduction of multiple genes is relatively straightforward in
that operons can be constructed to express multiple open reading
frames, or multiple complete genes can be expressed from a single
plasmid. However, introduction of pathways into plants is more
difficult due in part to the complexity of plant genes, the
difficulty of constructing vectors harboring multiple genes for
expression in plants, and the difficulty of introducing large
vectors intact into plants.
[0004] Polyhydroxyalkanoates are bacterial polyesters that
accumulate in a wide variety of bacteria. These polymers have
properties ranging from stiff and brittle plastics to rubber-like
materials, and are biodegradable. Because of these properties,
polyhydroxyalkanoates are an attractive source of non-polluting
plastics and elastomers.
[0005] Currently, there are approximately a dozen biodegradable
plastics in commercial use that possess properties suitable for
producing a number of specialty and commodity products (Lindsay,
Modern Plastics 2: 62, 1992). One such biodegradable plastic in the
polyhydroxyalkanoate (PHA) family that is commercially important is
Biopol.TM., a random copolymer of 3-hydroxybutyrate (3HB) and
3-hydroxyvalerate (3HV). This bioplastic is used to produce
biodegradable molded material (e.g., bottles), films, coatings, and
in drug release applications. Biopl.TM. is produced via a
fermentation process employing the bacterium Ralstonia eutropha
(Byrom, D. Trends Biotechnol. 5: 246-250, 1987). (R. eutropha was
formerly designated Alcaligenes eutrophus [Yabuuchi et al.,
Microbiol. Immunol. 39:897-904, 1995]). The current market price is
$6-7/lb, and the annual production is 1,000 tons. By best
estimates, this price can be reduced only about 2-fold via
fermentation (Poirier, Y. et al., Bio/Technology 13: 142, 1995).
Competitive synthetic plastics such as polypropylene and
polyethylene cost about 35-45.cent./lb (Layman, Chem. & Eng.
News, p. 10 (Oct. 31, 1994). The annual global demand for
polyethylene alone is about 37 million metric tons (Poirier, Y. et
al., Int. J. Biol. Macromol. 17: 7-12, 1995). It is therefore
likely that the cost of producing P(3HB-co-3HV) by microbial
fermentation will restrict its use to low-volume specialty
applications.
[0006] Polyhydroxyalkanoate (PHA) is a family of polymers composed
primarily of R-3-hydroxyalkanoic acids (Anderson, A. J. and Dawes,
E. A. Microbiol. Rev. 54: 450-472, 1990; Steinbuchel, A. in Novel
Biomaterials from Biological Sources, ed. Byrom, D. (MacMillan,
N.Y.), pp. 123-213, 1991); Poirier, Y., Nawrath, C. &
Somerville, C. Bio/Technology 13: 143-150, 1995).
Polyhydroxybutyrate (PHB) is the most well-characterized PHA. High
molecular weight PHB is found as intracellular inclusions in a wide
variety of bacteria (Steinbuchel, A. in Novel Biomaterials from
Biological Sources, ed. Byrom, D. (MacMillan, N.Y.), pp. 123-213,
1991). In Ralstonia eutropha, PHB typically accumulates to 80% dry
weight with inclusions being typically 0.2-1 .mu.m in diameter.
Small quantity of PHB oligomers of approximately 150 monomer units
are also found associated with membranes of bacteria and
eukaryotes, where they form channels permeable to calcium (Reusch,
R. N., Can. J. Microbiol. 41 (Suppl. 1): 50-54, 1995). High
molecular weight polyhydroxyalkanoates have the properties of
thermoplastics and elastomers. Numerous bacteria and fungi can
hydrolyze polyhydroxyalkanoates to monomers and oligomers, which
are metabolized as a carbon source. Polyhydroxyalkanoates have
accordingly attracted attention as a potential source of renewable
and biodegradable plastics and elastomers. PHB is a highly
crystalline polymer with rather poor physical properties, being
relatively stiff and brittle (de Koning, G., Can. J. Microbiol. 41
(Suppl. 1): 303-309, 1995). In contrast, PHA copolymers containing
monomer units ranging from 3 to 5 carbons for short-chain-length
PHA (SCL-PHA), or 6 to 14 carbons for medium-chain-length PHA
(MCL-PHA), are less crystalline and more flexible polymers (de
Koning, G., Can. J. Microbiol. 41 (Suppl. 1): 303-309, 1995).
[0007] PHB has been produced in the plant Arabidopsis thaliana
expressing the R. eutropha PHB biosynthetic enzymes (Poirier, Y. et
al., Science 256: 520-523, 1992; Nawrath, C., et al., Proc. Natl.
Acad. Sci. U.S.A. 91: 12760-12764, 1994). In plants expressing the
PHB pathway in the plastids, leaves accumulated up to 14% PHB per
gram dry weight (Nawrath, C., et al., Proc. Natl. Acad. Sci. U.S.A.
91: 12760-12764, 1994). High-level synthesis of PHB in plants
opened the possibility of utilizing agricultural crops as a
suitable system for the production of polyhydroxyalkanoates on a
large scale and at low cost (Poirier, Y. et al., Bio/Technology 13:
143-150, 1995; Poirier, Y. et al., FEMS Microbiol. Rev. 103:
237-246, 1992; Nawrath, C., et al. Molecular Breeding 1: 105-22,
1995). PHB was also shown to be synthesized in insect cells
expressing a mutant fatty acid synthase (Williams, M. D., et al.,
Appl. Environ. Microbiol. 62: 2540-2546, 1996), and in yeast
expressing the R. eutropha PHB synthase (Leaf, T. A., et al.
Microbiol. 142: 1169-1180, 1996).
[0008] A number of pseudomonads, including Pseudomonas putida and
Pseudomonas aeruginosa, accumulate MCL-PHAs when cells are grown on
alkanoic acids (Anderson, A. J. & Dawes, E. A. Microbiol. Rev.
54: 450-472, 1990; Steinbuchel, A. in Novel Biomaterials from
Biological Sources, ed. Byrom, D. (MacMillan, N.Y.), pp. 123-213,
1991; Poirier, Y., Nawrath, C. & Somerville, C. Bio/Technology
13: 143-150, 1995). The nature of the PHA produced is related to
the substrate used for growth and is typically composed of monomers
which are 2 n carbons shorter than the substrate. These studies
indicate that MCL-PHAs are synthesized by the PHA synthase from
3-hydroxyacyl-CoA intermediates generated by the .beta.-oxidation
of alkanoic acids (Huijberts, G. N. M., et al. Appl. Environ.
Microbiol. 58: 536-544, 1992; Huijberts, G. N. M., et al., J.
Bacteriol. 176: 1661-1666, 1994).
[0009] Chen et al. (Nature Biotech., 16: 1060-1064, 1998; reviewed
by Gelvin, S. B., Nature Biotech., 16: 1009-1010, 1998) describes
the cobombardment of embryogenic rice tissues with a mixture of 14
different pUC based plasmids. Integration of multiple transgenes
was observed to occur at one or two genetic loci.
[0010] Creating a transgenic host cell or plant that produces
multiple enzymes within a biosynthetic pathway is often a daunting
task. Individual vectors must be created for each enzyme.
Transformation of the host cell or plant is typically accomplished
by one of three general methods: serial transformation, parallel
transformation followed by crossing, or batch transformation. Each
method has serious practical drawbacks.
[0011] Serial transformation involves transforming a host cell or
plant with the first vector, selecting and characterizing the
transformed cell or plant, transforming with the second vector, and
so on. This process can become quite laborious and time
consuming.
[0012] Parallel transformation followed by crossing involves
separately transforming cells with each of the individual vectors,
and subsequently mating or crossbreeding the transformed cells or
plants to obtain a final cell or plant which contains all of the
individual sequences. This is a lengthy process, especially for the
crossbreeding of plant lines.
[0013] Batch transformation involves a single transformation event
involving all of the individual vectors. A wide array of cells are
produced, each containing between none and all of the vectors.
While only a single transformation is required, extensive
characterization of the resulting cells is necessary. As the number
of vectors increases, it is increasingly likely that no cells will
be obtained containing all of the vectors. If no desired
transformed cells are identified, the transformation must be
repeated.
[0014] An additional concern with all three of these methods is
that they do not allow any control over the relative copy numbers
of the individual vectors in the transformed cell or plant. It
would be desirable to have a transformation method that permits
control of the relative copy numbers of the individual sequences in
the transformed cell or plant, and also coordinates the positional
effect of the insertion locus.
[0015] There exists a need for improved materials and methods for
the preparation of transgenic organisms transformed with multiple
nucleic acid sequences encoding members of a multi-enzyme
biosynthetic pathway.
SUMMARY OF THE INVENTION
[0016] The invention involves the construction and use of nucleic
acid segments and vectors containing multiple sequences encoding
members of a biosynthetic pathway. The resulting vector allows a
single transformation event to produce a transformed cell or plant
containing all of the nucleic acid sequences. Furthermore, the
researcher has total control over the number of copies of each
coding sequence within the constructed vector. Single or multiple
copies of each coding sequence may easily be designed into the
vector.
[0017] An unexpected beneficial result of the invention is that
organisms transformed with a multi-enzyme coding vector produce the
biosynthetic product in higher yield than organisms produced by
serial transformation, parallel transformation with crossing, or
batch transformation methods.
DETAILED DESCRIPTION OF THE INVENTION
[0018] The invention is directed generally towards the construction
and use of nucleic acid segments comprising sequences encoding
multiple enzymes in a multi-enzyme biosynthetic pathway. The
biosynthetic pathway may generally be any biosynthetic pathway.
Examples of such multi-enzyme biosynthetic pathways are the TCA
cycle, polyketide synthesis pathway, carotenoid synthesis,
glycolysis, gluconeogenesis, starch synthesis, lignins and related
compounds, production of small molecules that serve as pesticides,
fungicides, or antibiotics, and polymer synthesis pathways.
Preferably, the biosynthetic pathway is a polyhydroxyalkanoate
biosynthesis pathway.
[0019] This disclosure describes multigene vectors designed to
produce polyhydroxyalkanoate (PHA) in plants. Some of these vectors
are designed to produce poly(.beta.-hydroxybutyrate), and some are
designed to produce
poly(.beta.-hydroxybutyrate-co-.beta.hydroxyvalerate) (Gruys et
al., WO 98/00557, 1998). In general, the efficiency of PHA
production was dramatically increased when all sequences necessay
for a pathway were introduced on the same vector. Herein,
construction of these multigene vectors, and their use for
polyhydroxyalkanoate production in Arabidopsis thaliana and
Brassica napus, and Zea mays is described.
[0020] An embodiment of the present invention is an isolated
nucleic acid segment comprising multiple nucleic acid sequences,
each encoding a different protein within the biosynthetic pathway.
Preferably, the isolated nucleic acid segment comprises a first
nucleic acid sequence encoding a polyhydroxyalkanoate synthase
protein; a second nucleic acid sequence encoding a .beta.-ketoacyl
reductase protein; and a third nucleic acid sequence encoding a
.beta.-ketothiolase protein. The nucleic acid segment may further
comprise additional nucleic acid sequences encoding additional
proteins such as a threonine deaminase protein or a deregulated
threonine deaminase protein.
[0021] An alternative embodiment of the invention is a recombinant
vector comprising multiple nucleic acid sequences, each encoding a
different protein within the biosynthetic pathway. The recombinant
vector may be arranged with a single promoter producing a
polycistronic RNA transcript from the multiple nucleic acid
sequences, or with each nucleic acid sequence being under the
control of its own promoter. The multiple promoters may be the same
or different. It is also possible to have one or more nucleic acid
sequence under the control of its own promoter, while other nucleic
acid sequences may be jointly under the control of a single
promoter producing a polycistronic RNA transcript.
[0022] A recombinant vector placing the biosynthetic pathway
nucleic acid sequences under the control of a single promoter
preferably comprises operatively linked in the 5' to 3' direction:
a promoter that directs transcription of the first nucleic acid
sequence, second nucleic acid sequence, and third nucleic acid
sequence; a first nucleic acid sequence; a second nucleic acid
sequence; a third nucleic acid sequence; a 3' transcription
terminator; and a 3' polyadenylation signal sequence; wherein: the
first nucleic acid sequence, second nucleic acid sequence, and
third nucleic acid sequence encode different proteins; and the
first nucleic acid sequence, second nucleic acid sequence, and
third nucleic acid sequence are independently selected from the
group consisting of a nucleic acid sequence encoding a
polyhydroxyalkanoate synthase protein, a nucleic acid sequence
encoding a .beta.-ketoacyl reductase protein, and a nucleic acid
sequence encoding a .beta.-ketothiolase protein. The nucleic acid
sequences encoding the biosynthetic pathway enzymes may be in any
order relative to each other and the promoter. The promoter must be
expressed in plastids. It may have either been derived from a
plastid, or may have been derived from a bacterium or phage having
promoters recognized by the plastid transcription enzymes, or be a
synthetic promoter recognized by the plastid transcription
enzymes.
[0023] A recombinant vector placing the biosynthetic pathway
nucleic acid sequences under the control of multiple promoters
preferably comprises a first element comprising operatively linked
in the 5' to 3' direction: a first promoter that directs
transcription of the first nucleic acid sequence; a first nucleic
acid sequence encoding a polyhydroxyalkanoate synthase protein; a
first 3' transcription terminator; a first 3' polyadenylation
signal sequence; a second element comprising operatively linked in
the 5' to 3' direction: a second promoter that directs
transcription of the second nucleic acid sequence; a second nucleic
acid sequence encoding a .beta.-ketoacyl reductase protein; a
second 3' transcription terminator; a second 3' polyadenylation
signal sequence; and a third element comprising operatively linked
in the 5' to 3' direction: a third promoter that directs
transcription of the third nucleic acid sequence; a third nucleic
acid sequence encoding a .beta.-ketothiolase protein; a third 3'
transcription terminator; and a third 3' polyadenylation signal
sequence. The .beta.-ketothiolase protein preferably condenses two
molecules of acetyl-CoA to produce acetoacetyl-CoA; and condenses
acetyl-CoA and propionyl-CoA to produce .beta.-ketovaleryl-CoA. The
.beta.-ketoacyl reductase protein preferably reduces
acetoacetyl-CoA to .beta.-hydroxybutyryl-CoA; and reduces
.beta.-ketovaleryl-CoA to .beta.-hydroxyvaleryl-CoA. The
polyhydroxyalkanoate synthase protein is preferably selected from
the group consisting of: a polyhydroxyalkanoate synthase protein
that incorporates .beta.-hydroxybutyryl-CoA into P(3HB) polymer;
and a polyhydroxyalkanoate synthase protein that incorporates a
.beta.-hydroxybutyryl-CoA and a .beta.-hydroxyvaleryl-CoA into
P(3HB-co-3HV) copolymer. The .beta.-ketothiolase protein may
comprise a transit peptide sequence that directs transport of the
.beta.-ketothiolase protein to the plastid. The .beta.-ketoacyl
reductase protein may comprise a transit peptide sequence that
directs transport of the .beta.-ketoacyl reductase protein to the
plastid. The polyhydroxyalkanoate synthase protein may comprise a
transit peptide sequence that directs transport of the
polyhydroxyalkanoate synthase protein to the plastid. The
recombinant vector may further comprise a nucleic acid sequence
encoding a threonine deaminase protein or a deregulated threonine
deaminase protein. The first promoter, second promoter, and third
promoter are preferably active in plants. The first promoter,
second promoter, and third promoter are preferably viral promoters.
The first promoter, second promoter, and third promoter are
preferably independently selected from the group consisting of a
CMV 35S promoter, an enhanced CMV 35S promoter, maize chlorophyll
A/B binding protein promoter, and an FMV 35S promoter. More
preferably, the first promoter, second promoter, and third promoter
are the CMV 35S promoter. The first promoter, second promoter, and
third promoter may be tissue specific promoters. The first
promoter, second promoter, and third promoter may independently be
the Lesquerella hydroxylase promoter or the 7S conglycinin
promoter, and preferably each is the Lesquerella hydroxylase
promoter.
[0024] An alternative embodiment is directed towards transformed
host cells. Transformed host cells may contain a non-integrated
recombinant vector or an integrated recombinant vector.
[0025] A transformed host cell may comprise a recombinant vector,
wherein the recombinant vector comprises a first element comprising
operatively linked in the 5' to 3' direction: a first promoter that
directs transcription of the first nucleic acid sequence; a first
nucleic acid sequence encoding a polyhydroxyalkanoate synthase
protein; a first 3' transcription terminator; a first 3'
polyadenylation signal sequence; a second element comprising
operatively linked in the 5' to 3' direction: a second promoter
that directs transcription of the second nucleic acid sequence; a
second nucleic acid sequence encoding a .beta.-ketoacyl reductase
protein; a second 3' transcription terminator; a second 3'
polyadenylation signal sequence; and a third element comprising
operatively linked in the 5' to 3' direction a third promoter that
directs transcription of the third nucleic acid sequence; a third
nucleic acid sequence encoding a .beta.-ketothiolase protein; a
third 3' transcription terminator; and a third 3' polyadenylation
signal sequence.
[0026] The transformed host cell may alternatively contain an
integrated nucleic acid segment. Preferably, the transformed host
cell may comprise a first element comprising operatively linked in
the 5' to 3' direction: a first promoter that directs transcription
of a first nucleic acid sequence; a first nucleic acid sequence
encoding a polyhydroxyalkanoate synthase protein; a first 3'
transcription terminator; a first 3' polyadenylation signal
sequence; a second element comprising operatively linked in the 5'
to 3' direction: a second promoter that directs transcription of a
second nucleic acid sequence; a second nucleic acid sequence
encoding a .beta.-ketoacyl reductase protein; a second 3'
transcription terminator; a second 3' polyadenylation signal
sequence; and a third element comprising operatively linked in the
5' to 3' direction: a third promoter that directs transcription of
a third nucleic acid sequence; a third nucleic acid sequence
encoding a .beta.-ketothiolase protein; a third 3' transcription
terminator; and a third 3' polyadenylation signal sequence. The
first element, second element, and third element may be
cointegrated within a continuous 10 Mb segment of genomic DNA, more
preferably within a continuous 5 Mb, 2.5 Mb, 2 Mb, 1.5 Mb, 1 Mb,
500 kb, 250 kb, 100 kb, 50 kb, or 20 kb segment of genomic DNA.
Alternatively, the first element, second element, and third element
may be cointegrated between a left Ti border sequence and a right
Ti border sequence. While it is preferable that a recombinant
vector contain a single left Ti border sequence and a single right
Ti border sequence, the invention encompasses recombinant vectors
containing multiple left and/or right Ti border sequences, and the
use thereof.
[0027] Alternatively, the host cell may comprise a nucleic acid
segment containing nucleic acid sequences encoding enzymes in a
biosynthetic pathway, where a single promoter directs transcription
of the nucleic acid sequences.
[0028] The transformed host cell may generally be any host cell,
and preferably is a bacterial, fungal, or plant cell. The bacterial
cell is preferably an Escherichia coli cell. The fungal cell is
preferably a yeast, Saccharomyces cerevisiae, or
Schizosaccharomyces pombe cell. The plant cell may be a monocot
plant cell, a dicot plant cell, an algae cell, or a conifer plant
cell. The plant cell is preferably a tobacco, wheat, potato,
Arabidopsis, corn, soybean, canola, sugar beet, oil seed rape,
sunflower, flax, peanut, sugarcane, switchgrass, or alfalfa
cell.
[0029] The promoters may be any of the promoters discussed earlier.
The transformed host cells preferably produce polyhydroxyalkanoate
polymer.
[0030] The invention also encompasses transformed plants. The
transformed plant may contain an integrated set of nucleic acid
sequences, or may contain the same set of nucleic acid sequences on
a non-integrated vector. A preferred embodiment is directed towards
a transformed plant comprising a first element comprising
operatively linked in the 5' to 3' direction: a first promoter that
directs transcription of a first nucleic acid sequence; a first
nucleic acid sequence encoding a polyhydroxyalkanoate synthase
protein; a first 3' transcription terminator; a first 3'
polyadenylation signal sequence; a second element comprising
operatively linked in the 5' to 3' direction: a second promoter
that directs transcription of a second nucleic acid sequence; a
second nucleic acid sequence encoding a .beta.-ketoacyl reductase
protein; a second 3' transcription terminator; a second 3'
polyadenylation signal sequence; and a third element comprising
operatively linked in the 5' to 3' direction: a third promoter that
directs transcription of a third nucleic acid sequence; a third
nucleic acid sequence encoding a .beta.-ketothiolase protein; a
third 3' transcription terminator; and a third 3' polyadenylation
signal sequence. The first element, second element, and third
element may be cointegrated within a continuous 10 Mb segment of
genomic DNA, more preferably within a continuous 5 Mb, 2.5 Mb, 2
Mb, 1.5 Mb, 1 Mb, 500 kb, 250 kb, 100 kb, 50 kb, or 20 kb segment
of genomic DNA. Alternatively, the first element, second element,
and third element may be cointegrated between a left Ti border
sequence and a right Ti border sequence.
[0031] Alternatively, the transformed plant may comprise a nucleic
acid segment containing nucleic acid sequences encoding enzymes in
a biosynthetic pathway, where a single promoter directs
transcription of the nucleic acid sequences.
[0032] The transformed plant may generally be any type of plant,
and preferably is a tobacco, wheat, potato, Arabidopsis, corn,
soybean, canola, oil seed rape, sunflower, flax, peanut, sugarcane,
switchgrass, or alfalfa plant.
[0033] The promoters may be any of the promoters discussed earlier.
The transformed plant preferably produces polyhydroxyalkanoate
polymer.
[0034] The invention also encompasses methods of preparing
transformed host cells. The methods may produce a transformed host
cell having nucleic acid sequences under the control of multiple
promoters or under the control of a single promoter. The method
preferably comprises the steps of selecting a host cell;
transforming the selected host cell with a recombinant vector
comprising: a first element comprising operatively linked in the 5'
to 3' direction: a first promoter that directs transcription of the
first nucleic acid sequence; a first nucleic acid sequence encoding
a polyhydroxyalkanoate synthase protein; a first 3' transcription
terminator; a first 3' polyadenylation signal sequence; a second
element comprising operatively linked in the 5' to 3' direction: a
second promoter that directs transcription of the second nucleic
acid sequence; a second nucleic acid sequence encoding a
.beta.-ketoacyl reductase protein; a second 3' transcription
terminator; a second 3' polyadenylation signal sequence; and a
third element comprising operatively linked in the 5' to 3'
direction: a third promoter that directs transcription of the third
nucleic acid sequence; a third nucleic acid sequence encoding a
.beta.-ketothiolase protein; a third 3' transcription terminator;
and a third 3' polyadenylation signal sequence; and obtaining
transformed host cells; wherein the transformed host cells produce
polyhydroxyalkanoate polymer.
[0035] Alternatively, the method of preparing transformed host
cells may comprise the steps of selecting a host cell; transforming
the selected host cell with a recombinant vector comprising
operatively linked in the 5' to 3' direction: a promoter that
directs transcription of a first nucleic acid sequence, second
nucleic acid sequence, and third nucleic acid sequence; a first
nucleic acid sequence; a second nucleic acid sequence; a third
nucleic acid sequence; a 3' transcription terminator; and a 3'
polyadenylation signal sequence; and obtaining transformed host
cells; wherein: the first nucleic acid sequence, second nucleic
acid sequence, and third nucleic acid sequence encode different
proteins; the first nucleic acid sequence, second nucleic acid
sequence, and third nucleic acid sequence are independently
selected from the group consisting of a nucleic acid sequence
encoding a polyhydroxyalkanoate synthase protein, a nucleic acid
sequence encoding a .beta.-ketoacyl reductase protein, and a
nucleic acid sequence encoding a .beta.-ketothiolase protein; and
the transformed host cells produce polyhydroxyalkanoate
polymer.
[0036] The promoters may be any of the promoters discussed
earlier.
[0037] Also disclosed are methods for preparing transformed plants.
The methods may produce a transformed plant having nucleic acid
sequences under the control of multiple promoters or under the
control of a single promoter. The method preferably comprises the
steps of selecting a host plant cell; transforming the selected
host plant cell with a recombinant vector comprising: a first
element comprising operatively linked in the 5' to 3' direction: a
first promoter that directs transcription of a first nucleic acid
sequence; a first nucleic acid sequence encoding a
polyhydroxyalkanoate synthase protein; a first 3' transcription
terminator; and a first 3' polyadenylation signal sequence; a
second element comprising operatively linked in the 5' to 3'
direction: a second promoter that directs transcription of a second
nucleic acid sequence; a second nucleic acid sequence encoding a
.beta.-ketoacyl reductase protein; a second 3' transcription
terminator; and a second 3' polyadenylation signal sequence; and a
third element comprising operatively linked in the 5' to 3'
direction: a third promoter that directs transcription of a third
nucleic acid sequence; a third nucleic acid sequence encoding a
.beta.-ketothiolase protein; a third 3' transcription terminator;
and a third 3' polyadenylation signal sequence; obtaining
transformed host plant cells; and regenerating the transformed host
plant cells to produce transformed plants, wherein the transformed
plants produce polyhydroxyalkanoate polymer.
[0038] Alternatively, the method of preparing a transformed plant
may comprise the steps of selecting a host plant cell; transforming
the selected host plant cell with a recombinant vector comprising
operatively linked in the 5' to 3' direction: a promoter that
directs transcription of a first nucleic acid sequence, second
nucleic acid sequence, and third nucleic acid sequence; a first
nucleic acid sequence; a second nucleic acid sequence; a third
nucleic acid sequence; a 3' transcription terminator; and a 3'
polyadenylation signal sequence; obtaining transformed host plant
cells; and regenerating the transformed host plant cells to produce
transformed plants; wherein: the first nucleic acid sequence,
second nucleic acid sequence, and third nucleic acid sequence
encode different proteins; the first nucleic acid sequence, second
nucleic acid sequence, and third nucleic acid sequence are
independently selected from the group consisting of a nucleic acid
sequence encoding a polyhydroxyalkanoate synthase protein, a
nucleic acid sequence encoding a .beta.-ketoacyl reductase protein,
and a nucleic acid sequence encoding a .beta.-ketothiolase protein;
and the transformed plants produce polyhydroxyalkanoate
polymer.
[0039] The promoters may be any of the promoters discussed
earlier.
[0040] The invention is also directed towards methods of producing
biomolecules of interest. The multiple enzymes in the biosynthetic
pathway may lead to the production of materials of commercial and
scientific interest. Preferably, the biomolecules are polymers, and
more preferably are polyhydroxyalkanoate polymers. The methods may
comprise obtaining any of the above described transformed host
cells or transformed plants, culturing or growing the transformed
host cells or transformed plants under conditions suitable for the
production of polyhydroxyalkanoate polymer, and recovering
polyhydroxyalkanoate polymer. The methods, may further comprise the
addition of nutrients, substrates, or other chemical additives to
the growth media or soil to facilitate production of
polyhydroxyalkanoate polymer. In a preferred embodiment, it is
possible to extract the polyhydroxyalkanoate from the transformed
host cells or transformed plants without killing the host cells or
plants. This may be accomplished, for example, by various solvent
extraction methods or by engineering the host cells or plants to
secrete the polyhydroxyalkanoate polymer, or by directing
production to tissues such as leaves or seeds which may be removed
without causing serious injury to the plant. The
polyhydroxyalkanoate polymer produced is preferably
poly(3-hydroxybutyrate), poly(3-hydroxybutyrate-co-3-hydroxyva-
lerate), poly(4-hydroxybutyrate), or
poly(3-hydroxybutyrate-co-4-hydroxybu- tyrate).
[0041] If repetitive sequences are used in a multi-gene plasmid
system, there exists the possibility for gene silencing in
subsequent generations of plants. If expression levels are high
gene silencing could also occur and would be independent of
repetitive elements. Repetitive sequences may include the use of
the same promoters, chloroplast peptide encoding sequences, and
other genetic elements for each of the multi-gene coding sequences.
Gene silencing often manifests itself as a gradual reduction in
protein levels, mRNA levels, or biosynthesis product concentrations
in subsequent generations of related plants. If gene silencing is
observed, changing the repetitive sequences through the use of
diverse genetic elements such as different promoters, leaders,
introns, transit peptide sequences, etc., different designed
nucleotide sequence, or through mutagenesis of the existing
sequence, may be successful in reducing or eliminating the gene
silencing effects.
DESCRIPTION OF THE FIGURES
[0042] The following figures form part of the present specification
and are included to further demonstrate certain aspects of the
present invention. The invention may be better understood by
reference to one or more of these drawings in combination with the
detailed description of specific embodiments presented herein.
[0043] FIG. 1: Biosynthesis of
poly(.beta.-hydroxybutyrate-co-.beta.-hydro- xyvalerate)
(poly(3HB-co-3HV), PHBV) in Ralstonia eutropha.
[0044] FIG. 2: Plant transformation strategies for multi-enzyme
metabolic pathway engineering.
[0045] FIG. 3: Plasmid map of pMON25642. A list of the restriction
enzyme cutting sites for pMON25642 is provided in Table 10.
[0046] FIG. 4: Plasmid map of pMON10098. A list of the restriction
enzyme cutting sites for pMON10098 is provided in Table 11.
[0047] FIG. 5: Plasmid map of pMON969. A list of the restriction
enzyme cutting sites for pMON969 is provided in Table 12.
[0048] FIG. 6: Plasmid map of pMON25661. A list of the restriction
enzyme cutting sites for pMON25661 is provided in Table 13.
[0049] FIG. 7: Plasmid map of pMON25897. A list of the restriction
enzyme cutting sites for pMON25897 is provided in Table 14.
[0050] FIG. 8: Plasmid map of pMON25662. A list of the restriction
enzyme cutting sites for pMON25662 is provided in Table 15.
[0051] FIG. 9: Plasmid map of pMON25663. A list of the restriction
enzyme cutting sites for pMON25663 is provided in Table 16.
[0052] FIG. 10: Plasmid map of pMON25943. A list of the restriction
enzyme cutting sites for pMON25943 is provided in Table 17.
[0053] FIG. 11: Plasmid map of pMON25948. A list of the restriction
enzyme cutting sites for pMON25948 is provided in Table 18.
[0054] FIG. 12: Plasmid map of pMON25949. A list of the restriction
enzyme cutting sites for pMON25949 is provided in Table 19.
[0055] FIG. 13: Plasmid map of pMON25951. A list of the restriction
enzyme cutting sites for pMON25951 is provided in Table 20.
[0056] FIG. 14: Plasmid map of pMON34545. A list of the restriction
enzyme cutting sites for pMON34545 is provided in Table 21.
[0057] FIG. 15: Plasmid map of pMON34565. A list of the restriction
enzyme cutting sites for pMON34565 is provided in Table 22.
[0058] FIG. 16: Plasmid map of pMON25995. A list of the restriction
enzyme cutting sites for pMON25995 is provided in Table 23.
[0059] FIG. 17: Plasmid map of pMON25973. A list of the restriction
enzyme cutting sites for pMON25973 is provided in Table 24.
[0060] FIG. 18: Plasmid map of pMON25987. A list of the restriction
enzyme cutting sites for pMON25987 is provided in Table 25.
[0061] FIG. 19: Plasmid map of pMON25991. A list of the restriction
enzyme cutting sites for pMON25991 is provided in Table 26.
[0062] FIG. 20: Plasmid map of pMON25992. A list of the restriction
enzyme cutting sites for pMON25992 is provided in Table 27.
[0063] FIG. 21: Plasmid map of pMON25993. A list of the restriction
enzyme cutting sites for pMON25993 is provided in Table 28.
[0064] FIG. 22: Plasmid map of pMON36805. A list of the restriction
enzyme cutting sites for pMON36805 is provided in Table 29.
[0065] FIG. 23: Plasmid map of pMON36814. A list of the restriction
enzyme cutting sites for pMON36814 is provided in Table 30.
[0066] FIG. 24: Plasmid map of pMON36816. A list of the restriction
enzyme cutting sites for pMON36816 is provided in Table 31.
[0067] FIG. 25: Plasmid map of pMON36824. A list of the restriction
enzyme cutting sites for pMON36824 is provided in Table 32.
[0068] FIG. 26: Plasmid map of pMON36843. A list of the restriction
enzyme cutting sites for pMON36843 is provided in Table 33.
[0069] FIG. 27: Plasmid map of pMON34543. A list of the restriction
enzyme cutting sites for pMON34543 is provided in Table 34.
[0070] FIG. 28: Plasmid map of pMON36850. A list of the restriction
enzyme cutting sites for pMON36850 is provided in Table 35.
[0071] FIG. 29: Plasmid map of pMON25963. A list of the restriction
enzyme cutting sites for pMON25963 is provided in Table 36.
[0072] FIG. 30: Plasmid map of pMON25965. A list of the restriction
enzyme cutting sites for pMON25965 is provided in Table 37.
[0073] FIG. 31: Method for creating multi-gene vectors.
[0074] FIG. 32: PHB biosynthetic pathway. PHB production requires
the condensation of two acetyl-CoA molecules using a
.beta.-ketothiolase, a D-isomer-specific reduction by
acetoacetyl-CoA reductase, and PHB polymerization by PHB synthase.
The genes encoding these enzymes are indicated in parentheses.
[0075] FIG. 33: Schematic diagram of multi-gene vector used to
transform Brassica napus. Vectors were constructed using modular
cassettes. Each cassette consists of the Lesquerella hydroxylase
promoter (P-Lh), a chloroplast transit peptide (ctp) fused to an
open reading frame encoding a PHB synthesis enzyme, and the E9 3'
terminator. The plasmid also expresses EPSP synthase to provide
resistance to glyphosate, contains bacterial replication origins,
and a bacterially-expressed gene encoding resistance to
streptomycin and spectinomycin. In pMON36814, bktB was replaced
with phbA. Otherwise, the vectors were identical. RB, right border
of T-DNA; LB, left border of T-DNA.
[0076] FIG. 34: Electron micrographs of Brassica napus plastids.
Panel A: Leukoplast from wild type Brassica napus seed. Panel B:
Leukoplast from Brassica napus seed producing PHB. Polymer (PHB)
and oil bodies (0) are indicated. Note the greatly expanded size of
leukoplasts in the PHB-producing line.
[0077] FIG. 35: A pathway designed to produce
poly(P-hydroxybutyrate-co-.b- eta.-hydroxyvalerate) in the plastids
of plants. Propionyl-CoA is derived from threonine via threonine
deaminase and the pyruvate dehydrogenase complex. Acetyl-CoA is
drawn from normal intermediary metabolism. The pathway requires
transformation of the plant with four genes (ilvA, bktB, phbB, and
phbC), and relies on endogenous pyruvate dehydrogenase. All enzymes
encoded by transgenes are targeted to the plastid using chloroplast
transit peptides.
[0078] FIG. 36: Concentrations of selected 2-keto acids and amino
acids in control plants and in Arabidopsis expressing threonine
deaminase. (A) Comparison of pyruvate and 2-ketobutyrate
concentrations in Arabidopsis harboring either a control plasmid or
a plasmid expressing wild type E. coli ilvA (threonine deaminase).
(B) Comparison of threonine, isoleucine, and 2-ketobutyrate
concentrations in Arabidopsis harboring either a control plasmid or
a plasmid expressing wild type E. coli ilvA. Note the different
scales used in parts (A) and (B).
[0079] FIG. 37: .sup.3C NMR spectra demonstrating
poly(P-hydroxybutyrate-c- o-.beta.-hydroxyvalerate) copolymer
production in transgenic Arabidopsis. Note the presence of signals
indicating presence of both 3-hydroxybutyrate and 3-hydroxyvalerate
side chains.
[0080] FIG. 38: Analyses of total polymer production, the
3-hydroxyvalerate fraction of the polymer, and the activity of
threonine deaminase Brassica oilseeds synthesizing PHBV copolymer.
Note the distinct negative correlation between polymer
concentration and the 3-HV content of the polymer. Also note that
increasing threonine deaminase activity does not lead to increased
3-HV content.
[0081] FIG. 39: Multiple potential routes to produce propionyl-CoA
in planta. Most alternative pathways have the potential to produce
propionyl-CoA in plants. However, production of propionyl-CoA from
threonine provides the most direct route.
[0082] FIG. 40: Bar graph of average % PHA produced from
Arabidopsis transformation methods.
[0083] FIG. 41: Bar graph of average % PHA produced from canola
transformation methods.
[0084] FIG. 42: Bar graph of maximum % PHA produced from
Arabidopsis transformation methods.
[0085] FIG. 43: Bar graph of maximum % PHA produced from canola
transformation methods.
DEFINITIONS
[0086] The following definitions are provided in order to aid those
skilled in the art in understanding the detailed description of the
present invention.
[0087] "Acyl-ACP thioesterase" refers to proteins which catalyze
the hydrolysis of acyl-ACP thioesters.
[0088] "C-terminal region" refers to the region of a peptide,
polypeptide, or protein chain from the middle thereof to the end
that carries the amino acid having a free a carboxyl group (the
C-terminus).
[0089] "CoA" refers to coenzyme A.
[0090] The phrases "coding sequence", "open reading frame", and
"structural sequence" refer to the region of continuous sequential
nucleic acid triplets encoding a protein, polypeptide, or peptide
sequence.
[0091] The term "encoding DNA" or "encoding nucleic acid" refers to
chromosomal nucleic acid, plasmid nucleic acid, cDNA, or synthetic
nucleic acid which codes on expression for any of the proteins or
fusion proteins discussed herein.
[0092] "Fatty acyl hydroxylase" refers to proteins which catalyze
the conversion of fatty acids to hydroxylated fatty acids.
[0093] The term "genome" as it applies to bacteria encompasses both
the chromosome and plasmids within a bacterial host cell. Encoding
nucleic acids of the present invention introduced into bacterial
host cells can therefore be either chromosomally-integrated or
plasmid-localized. The term "genome" as it applies to plant cells
encompasses not only chromosomal DNA found within the nucleus, but
organelle DNA found within subcellular components of the cell.
Nucleic acids of the present invention introduced into plant cells
can therefore be either chromosomally-integrated or
organelle-localized.
[0094] "Identity" refers to the degree of similarity between two
nucleic acid or protein sequences. An alignment of the two
sequences is performed by a suitable computer program. A widely
used and accepted computer program for performing sequence
alignments is CLUSTALW v1.6 (Thompson, et al. Nucl. Acids Res., 22:
4673-4680, 1994). The number of matching bases or amino acids is
divided by the total number of bases or amino acids, and multiplied
by 100 to obtain a percent identity. For example, if two 580 base
pair sequences had 145 matched bases, they would be 25 percent
identical. If the two compared sequences are of different lengths,
the number of matches is divided by the shorter of the two lengths.
For example, if there were 100 matched amino acids between 200 and
a 400 amino acid proteins, they are 50 percent identical with
respect to the shorter sequence. If the shorter sequence is less
than 150 bases or 50 amino acids in length, the number of matches
are divided by 150 (for nucleic acids) or 50 (for proteins); and
multiplied by 100 to obtain a percent identity.
[0095] The terms "microbe" or "microorganism" refer to algae,
bacteria, fungi, and protozoa.
[0096] "N-terminal region" refers to the region of a peptide,
polypeptide, or protein chain from the amino acid having a free a
amino group to the middle of the chain.
[0097] "Nucleic acid" refers to ribonucleic acid (RNA) and
deoxyribonucleic acid (DNA).
[0098] A "nucleic acid segment" is a nucleic acid molecule that has
been isolated free of total genomic DNA of a particular species, or
that has been synthesized. Included with the term "nucleic acid
segment" are DNA segments, recombinant vectors, plasmids, cosmids,
phagemids, phage, viruses, etcetera.
[0099] "Overexpression" refers to the expression of a polypeptide
or protein encoded by a DNA introduced into a host cell, wherein
said polypeptide or protein is either not normally present in the
host cell, or wherein said polypeptide or protein is present in
said host cell at a higher level than that normally expressed from
the endogenous gene encoding said polypeptide or protein.
[0100] The term "plastid" refers to the class of plant cell
organelles that includes amyloplasts, chloroplasts, chromoplasts,
elaioplasts, eoplasts, etioplasts, leucoplasts, and proplastids.
These organelles are self-replicating, and contain what is commonly
referred to as the "chloroplast genome," a circular DNA molecule
that ranges in size from about 120 to about 217 kb, depending upon
the plant species, and which usually contains an inverted repeat
region (Fosket, Plant growth and Development, Academic Press, Inc.,
San Diego, Calif., p. 132, 1994).
[0101] "Polyadenylation signal" or "polyA signal" refers to a
nucleic acid sequence located 3' to a coding region that directs
the addition of adenylate nuclecotides to the 3' end of the mRNA
transcribed from the coding region.
[0102] The term "polyhydroxyalkanoate (or PHA) synthase" refers to
enzymes that convert hydroxyacyl-CoAs to polyhydroxyalkanoates and
free CoA.
[0103] The term "promoter" or "promoter region" refers to a nucleic
acid sequence, usually found upstream (5') to a coding sequence,
that controls expression of the coding sequence by controlling
production of messenger RNA (mRNA) by providing the recognition
site for RNA polymerase and/or other factors necessary for start of
transcription at the correct site. As contemplated herein, a
promoter or promoter region includes variations of promoters
derived by means of ligation to various regulatory sequences,
random or controlled mutagenesis, and addition or duplication of
enhancer sequences. The promoter region disclosed herein, and
biologically functional equivalents thereof, are responsible for
driving the transcription of coding sequences under their control
when introduced into a host as part of a suitable recombinant
vector, as demonstrated by its ability to produce mRNA.
[0104] "Regeneration" refers to the process of growing a plant from
a plant cell (e.g., plant protoplast or explant).
[0105] "Transformation" refers to a process of introducing an
exogenous nucleic acid sequence (e.g., a vector, recombinant
nucleic acid molecule) into a cell or protoplast in which that
exogenous nucleic acid is incorporated into a chromosome or is
capable of autonomous replication.
[0106] A "transformed cell" is a cell whose nucleic acid has been
altered by the introduction of an exogenous nucleic acid molecule
into that cell.
[0107] A "transformed plant" or "transgenic plant" is a plant whose
nucleic acid has been altered by the introduction of an exogenous
nucleic acid molecule into that plant, or by the introduction of an
exogenous nucleic acid molecule into a plant cell from which the
plant was regenerated or derived.
[0108] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventors to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
EXAMPLES
Example 1
Sources of Nucleic Acid Sequences
[0109] Nucleic acid sequences encoding the polyhydroxyalkanoate
biosynthetic pathway include: phbA and phbB (GenBank accession
number J04987), phbC (GenBank accession number J05003), and bktB
(GenBank accession number AF026544). Production of PHBV copolymer
can be accomplished by also expressing E. coli ilvA (GenBank
accession number U00096, overlapping base 3953951:Gruys et al. WO
98/00557). The Ti DNA left border sequence is described in Baker,
R. F., et al. (Plant Mol. Biol., 2: 335-350, 1983). The Ti DNA
right border sequence is described in Depicker et. al. (J. Mol.
App. Genet. 1: 561, 1982).
Example 2
Analysis of Nawrath Arabidopsis plants
[0110] Polyhydroxyalkanoates are a form of polyester accumulated by
numerous bacterial species as a carbon and energy repository. This
class of polymer also has useful thermoplastic properties, and is
therefore of interest as a biodegradable plastic.
Poly(.beta.-hydroxybutyrate-co-.beta- .-hydroxyvalerate)
(poly(3HB-co-3HV), PHBV), a form of PHA, is commercially produced
via fermentation of Ralstonia eutropha (FIG. 1). However, it is
expected that the cost of production could be dramatically
decreased if PHA could be produced in transgenic plants. The first
attempts at PHA production in plants utilized transgenic
Arabidopsis expressing the three genes required for the homopolymer
poly-.beta.-hydroxybutyrate (PHB) (Nawrath, C. et al., Proc. Natl.
Acad. Sci. U.S.A. 91: 12760-12764, 1994). In this work, the authors
transformed Arabidopsis plants with three independent gene
cassettes and crossed the plants using traditional breeding
methods. They reported PHB production up to 14% of the cell dry
weight. However, this method took a significant amount of time
before the three gene pathway could be assembled. In addition, the
plants did not maintain a stable phb.sup.+ phenotype, as determined
by our analysis of the progeny of these original plants (Table 1).
This problem may be due to co-suppression (Finnegan, J., and D.
McElroy. Bio/Technology. 12: 883-888, 1994), or to segregation of
high-producing insertions in the progeny. The plants produced by
Nawrath et al. were not fully characterized genetically, although
it is known that all contained multiple insertions of the
transgenes.
1TABLE 1 Enzyme activity and polymer data of progeny of Nawrath
Arabidopsis lines. Specific activities Western results % plant line
[protein] thiolase reductase PhbA PhbB PhbC polymer number (mg/mL)
(u/mg) (u/mg) thiolase reductase synthase (C4) 134 0.158 0.027
0.069 + + - 0.041% 140 0.189 0.026 0.019 + + - 0.068% 151 0.377
0.042 0.045 + + + 0.038% 159 0.127 0.025 0.009 - - + 0.053% 168
0.216 0.018 0.034 + + + 0.070% 175 0.186 0.010 0.028 + - - 0.043%
177 0.166 0.026 0.000 + - - 0.043% 203 0.144 0.030 0.043 - + +
0.034% 228 0.250 0.038 0.021 + + + 0.048% 240 0.192 0.023 0.010 NA
NA NA 0.045%
Example 3
Use of Multiple Vectors to Introduce PHA Biosynthesis Sequences
into Arabidopsis
[0111] One vector was constructed containing sequences encoding
both acetoacetyl-CoA reductase and PHB synthase proteins. A second
vector was constructed containing a sequence encoding a
.beta.-ketothiolase protein. Two independent transformation events
were obtained corresponding to each of these vectors. The complete
pathway was assembled into a single plant using traditional
cross-breeding methods. In all cases, plants exhibiting Mendelian
segregation consistent with transgene insertion at a single locus
were chosen. The results of these experiments are shown in Table
2.
[0112] The second strategy pursued was to simultaneously
co-transform both plasmids into a single plant (simultaneous
co-transformation) and assay the primary transformant for polymer
accumulation, or to re-transform plants that already harbored a
single vector (serial co-transformation). The results of these
experiments are summarized in Table 3. Although the activity of
enzymes expressed from the encoding sequences was comparable to
that reported by Nawrath et al., none of the plants generated
reached the polymer levels reportedly achieved in their study.
Neither their experiments nor these results correlate enzyme
activity with the intracellular concentration of PHA polymer
(Nawrath, C. et al., Proc. Natl. Acad. Sci. U.S.A. 91: 12760-12764,
1994).
2TABLE 2 Polymer data for Arabidopsis crosses. Vector Plant
construct # of lines # of lines C4 polymer Number description
assayed positive (% cell dry wt.) 25640 e35s ctpl phbA 0.01-1.55%
25665 e35s ctpl phbC 11 10 AVE: 0.651% e35s ctpl phbB SD: 0.596%
25640 e35s ctpl phbA 0.03-0.047% 25739 e35s ctpl phbB 20 12 AVE:
0.178% e35s ctpl nocC SD: 0.163% 25785 e35s ctpl bktB 0.04-0.88%
25665 e35s ctpl phbC 11 11 AVE: 0.354% e35s ctpl phbB SD: 0.199%
25785 e35s ctpl bktB 0.03-0.21% 25739 e35s ctpl phbB 24 9 AVE:
0.065% e35s ctpl nocC SD: 0.053% 25801 e35s ctpl bktB 0.02-0.04%
e35s ctpl ilvA466 8 3 AVE: 0.029% 25665 e35s ctpl phbC SD: 0.0095%
e35s ctpl phbB 25801 e35s ctpl bktB 0.03-0.091% e35s ctpl ilvA466
17 9 AVE: 0.044% 25739 e35s ctpl phbB SD: 0.022% e35s ctpl nocC
25812 e35s ctpl bktB 0.03-0.102% e35s ctpl ilvA w.t. 3 3 AVE:
0.073% 25665 e35s ctpl phbC SD: 0.035% e35s ctpl phbB 25812 e35s
ctpl bktB 0.02-0.11% e35s ctpl ilvA w.t. 10 7 AVE: 0.064% 25739
e35s ctpl phbB SD: 0.031% e35s ctpl nocC 64/104 plants positive;
AVE = average; SD = standard deviation.
[0113]
3TABLE 3 Polymer data for re-transformed and co-transformed
Arabidopsis. Vector Plant construct # of lines # of lines C4
polymer Number description assayed positive (% cell dry wt.) 25665
e35s ctpl phbC 0.03-0.81% e35s ctpl phbB 14 6 AVE: 0.25% RE/25880
e35s ctpl bktB SD: 0.29% e35s ctpl ilvA w.t. 25665 e35s ctpl phbC
e35s ctpl phbB 5 0 NA RE/25881 e35s ctpl bktB e35s ctpl ilvA219
25665 e35s ctpl phbC 0.02-0.33% e35s ctpl phbB 23 4 AVE: 0.16%
RE/25882 e35s ctpl bktB SD: 1.3% e35s ctpl ilvA466 25785 e35s ctpl
bktB 0.02-1.67% 25678 e35s ctpl phbB 21 8 AVE: 0.50% e35s ctpl phbC
SD: 0.64% 25785 e35s ctpl bktB 0.01-0.72% 25740 e35s ctpl phbB 27
18 AVE: 0.11 e35s ctpl nocC SD: 0.15 25801 e35s ctpl bktB
0.646-0.715% e35s ctpl ilvA466 2 1 AVE: 0.681 25678 e35s ctpl phbB
SD: 0.049% e35s ctpl phbC 25801 e35s ctpl bktB 0.02-0.17 e35s ctpl
ilvA466 28 16 AVE: 0.083% 25740 e35s ctpl phbB SD: 0.050% e35s ctpl
nocC 25812 e35s ctpl bktB 0.63-1.65% e35s ctpl ilvA w.t. 3 3* AVE:
1.191% 25678 e35s ctpl phbB SD: 0.463% e35s ctpl phbC 25812 e35s
ctpl bktB 0.02-0.20% e35s ctpl ilvA w.t. 30 9 AVE: 0.112% 25740
e35s ctpl phbB SD: 0.053% e35s ctpl nocC 64/145 plants positive. RE
indicates that this vector was used to re-transform a plant line.
AVE = average. SD = standard deviation.
Example 4
Construction of Multigene Vectors for Transformation of
Arabidopsis
[0114] In an attempt to increase the speed and simplicity of
genetic analysis, multigene vectors were constructed containing the
entire PHB biosynthetic pathway on a single plasmid. Multigene
vectors for PHA production in Arabidopsis were constructed from a
series of base vectors, each with the desired open reading frame
under control of the e35s promoter (Odell, J. T., et al, Nature,
313: 810-812, 1985) and the E9 3' region (Coruzzi, EMBO J.
3:1671-1679, 1984). The first vector in this series, pMON25642
(FIG. 3), harbors phbC under control of the e35s promoter in
pMON10098 (FIG. 4), a vector designed for Agrobacterium-mediated
transformation of plants. The remaining intermediate vectors are
all derived from pMON969 (FIG. 5), a high copy-number vector
harboring the e35s promoter and the E9 3' region. Constructs
derived from pMON969 include those encoding phbA (pMON25661; FIG.
6), bktB (pMON25897; FIG. 7), phbB (pMON25662; FIG. 8), and ilvA
(pMON25663; FIG. 9). From these and similar vectors were derived
the final plasmids for transformation of Arabidopsis; pMON25943
(FIG. 10) pMON25948 (FIG. 11), pMON25949 (FIG. 12), pMON25951 (FIG.
13), and pMON34545 (FIG. 14). All cloning procedures were performed
using standard ligation techniques (Sambrook, J., et al, "Molecular
cloning: A laboratory manual," Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., 1989), except that ligation of NotI-cut
pMON25949 with the ilvA-containing NotI restriction fragment of
pMON25663 produced plasmid pMON34565 (FIG. 15), that
serendipitously contained two copies of the ilvA fragment. Each
copy of ilvA contains a SnaBI restriction site, so deletion of a
3155 bp SnaBI restriction fragment from pMON34565 produced plasmid
pMON34545, a plasmid with a single copy of ilvA.
[0115] The final vectors, pMON25943, pMON25948, pMON25949,
pMON25951, and pMON34545 were used for Agrobacterium-mediated
transformation of Arabidopsis (Bechtold N., et al. Comptes Rendus
Acad. Sci. Paris Sciences Serie III Sciences de la Vie. 94-1199,
1993). This approach has proven successful in generating lines with
the highest levels of PHB obtained to date in our laboratory. PHA
production in the planats resulting from the first four of these
vectors is summarized in Table 4. Data from pMON34545
transformations will be obtained. All of the data in Table 4 were
derived from heterozygous plants, and the polymer concentration may
increase once the plants are brought to homozygosity. For example,
one plant that produced about 7% PHB by dry weight when
heterozygous produced polymer up to 13% when homozygous.
4TABLE 4 Polymer results from Arabidopsis derived from multigene
vectors. Plant construct # of lines # of lines C4 polymer Vector
number description assayed positive (% cell dry wt.) e35s ctpl phbC
0.11-2.94% 25943 e35s ctp2 phbB 34 28 AVE: 1.13% e35s ctpl bktB SD:
0.65% e35s ctpl phbC 0.01-7.63% 25948 e35s etpl phbA 53 46 AVE:
2.08% e35s etpl phbB SD: 1.56% e35s ctpl phbC 0.02-7.74% 25949 e35s
ctp2 bktB 35 30 AVE: 1.82% e35s ctpl phbB SD: 1.39% e35s ctpl phbC
0.20-3.78% 25951 e35s ctpl bktB 12 11 AVE: 1.60% e35s ctpl phbB SD:
1.04% 153/172 plants positive for PHB; 7 had greater than 4% dry
weight; AVE = average; SD = standard deviation
[0116] These results demonstrate that use of a multigene vector
provides consistently higher levels of polymer production than were
achieved using multiple vectors. The striking beneficial results in
polymer production obtained from the use of multigene vectors are
visually displayed in FIGS. 40 and 42.
[0117] There are several possible explanations for the increased
levels of polymer present in the multigene vector transformants.
One explanation derives from the fact that it was possible to
generate more independent lines with the multigene vectors, and the
screening of more plants allowed detection of the relatively rare
high-producing lines. This is one clear advantage of having the
entire pathway on a single vector, but the distribution of polymer
production in plants produced by the various methods suggests that
numbers alone do not account for the increased polymer production
of multigene vectors. It is also possible that having a metabolic
pathway genetically linked at a single integration locus is more
metabolically favorable due to some level of concerted gene
expression and/or mRNA metabolism. This phenomenon is common in
bacteria, but there are not many examples of clustering genes in
plants for concerted gene expression. Another possibility is that
the high local concentration of promoters may lead to locally high
levels of transcription factors. Still another possibility is that
having the genes tightly linked may reduce gene silencing, or
co-suppression, in certain cases.
Example 5
Extraction of Polymer from Arabidopsis and Analysis of Polymer
[0118] For isolation of polymer from Arabidopsis, stems and leaves
were harvested and dehydrated by lyophilization for approximately
36 hours. The material was ground to a fine powder, and 100 mg of
powder was treated with 10 mL CLOROX bleach (CLOROX is a registered
trademark of The Clorox Company, Oakland, Calif.) for 1 hour with
shaking at room temperature. The extract was subjected to
centrifugation at 2700.times. g for 10 minutes at 4.degree. C., and
the supernatant solutions was carefully removed. Ten mL 100%
methanol were added, the solution was mixed by vortexing, and then
centrifuged again. After a second, identical methanol extraction,
the material was allowed to dry overnight. Polymer was extracted
from the dried material with 1 mL of chloroform containing 1
.mu.mol/mL methyl-benzoate standard. The tube was heated to
100.degree. C. for 2.5 hours, solid material was removed by
centrifugation, and the supernatant material was subjected to
methanolysis. Methanolysis of polymer and gas chromatographic
characterization of the methyl-ester residues were performed as
described by Slater et al. (J. Bacteriol. 180:1979-1987, 1998).
Example 6
Use of Multiple Vectors for Gene Expression in the Seeds of
Canola
[0119] Production of polyhydroxyalkanoate has also been
accomplished within the seed of canola (oil seed rape). Initial
efforts followed essentially the same strategy as the initial
Arabidopsis strategy. That is, one vector carried the sequences
encoding acetoacetyl-CoA reductase and PHA synthase proteins, while
another carried the sequence encoding a .beta.-ketothiolase
protein. However the 7s promoter, which is expressed primarily in
the seed, replaced the 35s promoter that was used in the
Arabidopsis constructs. These 7s promoter vectors were used to
transform oilseed rape, homozygous lines were crossed, and PHB
accumulation was assayed in the resulting lines (Table 5). A number
of lines that produce PHB were identified, but all produced
relatively low concentrations of polymer, with the best lines
containing about 2% polymer by dry weight.
5TABLE 5 Polymer results for canola crosses. Plant construct # of
plants # of plants C4 polymer Vector number description assayed
positive (% dry wt.) 25638 7s ctpl phbA 0.024-1.99% 25626 7s ctpl
phbC 42 37 0.58% 7s ctpl phbB SD: 0.59% 25638 7s ctpl phbA
0.039-0.053 25741 7s tpss phbC 12 2 0.05% 7s tpss phbB SD: 0.01%
25818 7s ctpl bktB 0.04-1.67% 7s ctpl ilvA w.t. 22 17 AVE: 0.61%
25626 7s ctpl phbC SD: 0.43% 7s ctpl phbB 25818 7s ctpl bktB 7s
ctpl ilvA w.t. 15 0 NA 25741 7s tpss phbC 7s tpss phbB 25820 7s
ctpl bktB 0.26-0.72% 7s ctpl ilvA466 19 12 AVE: 0.51% 25626 7s ctpl
phbC SD: 0.16% 7s ctpl phbB 25820 7s ctpl bktB 7s ctpl ilvA466 7 0
NA 25741 7s tpss phbC 7s tpss phbB
Example 7
Construction of Multigene Vectors for Transformation of Canola
[0120] Large vectors for expression of multiple genes have also
been used to produce polyhydroxyalkanoate in the seeds of canola
(oil seed rape). In this case, the promoter was derived from the
fatty acid hydroxylase gene of Lesquerella (P-lh) (Broun, P. and C.
Somerville. Plant Physiol. 113: 933-942, 1997), which is expressed
primarily within the developing seed. A series of vectors, each
expressing the entire PHA biosynthesis pathway, was used for
transformation of oilseed rape. The multigene vectors were
constructed from a series of base vectors, each with the desired
open reading frame under control of the Lesquerella hydroxylase
promoter (P-lh; Broun, P. and Somerville, C. R. Plant Physiol.,
113: 933-942, 1987) and the E9 3' region. The first vector in this
series, pMON25995 (FIG. 16), harbors phbC under control of P-lh in
pMON25973 (FIG. 17), a vector designed for Agrobacterium-mediated
transformation of plants. The remaining intermediate vectors are
all derived from pMON25987 (FIG. 18), a high copy-number vector
harboring P-lh and the E9 3' region. Constructs derived from
pMON25987 (FIG. 16) include those encoding phbA (pMON25991; FIG.
19), bktB (pMON25992; FIG. 20), phbB (pMON25993; FIG. 21), and ilvA
(pMON36805; FIG. 22). These intermediate vectors were used to
construct the final vectors for oilseed rape transformation;
pMON36814 (FIG. 23), pMON36816 (FIG. 24), and pMON36824 (FIG.
25).
[0121] Construction of the multigene vectors for oilseed rape was
not as straightforward as was the construction of the Arabidopsis
vectors. This was primarily due to the large size of the promoter
(P-lh is about 2.2 kb), and the resulting larger size of the
multigene vector intermediates. As the vectors increased in size,
it was found to be most efficient to perform ligations of two
similar sized fragments, rather than one large vector and one small
incoming fragment. In addition, it was desirable to avoid partial
digests of the large vectors, and to perform cloning in which
opposite ends of an individual fragment were not compatible. A
number of intermediate vectors were constructed specifically to
allow cloning in this manner. Another advantage of this approach is
that it often allowed restriction enzyme-mediated digestion of the
parental plasmids prior to transformation of Escherichia coli with
ligation products. This procedure significantly increased the
frequency of correct constructs recovered. The final vectors were
used for Agrobacterium-mediated transformation of oilseed rape
(Fry, J. et al., Plant Cell Rep. 6: 321-325, 1987).
[0122] The results of oilseed rape transformation with the
multigene vectors are shown in Table 6. There are two primary
points of interest in these data. First, multigene vectors larger
than 26 kb were successfully constructed and used to transform
oilseed rape, with a very low percentage of the plants failing to
produce polymer. Second, the distribution of polymer concentrations
among multigene vector transformants is higher than that of the
plants derived from two separate 7s vectors.
6TABLE 6 Polymer results from canola transformed with multigene
vectors. Plant construct # of plants # of plants C4 polymer Vector
number description assayed positive (% dry wt.) 36814 lhydrox ctpl
phbC 0.19-4.11% lhydrox ctpl phbA 68 59 AVE: 1.43% lhydrox tpss
phbB SD: 1.01% 36816 lhydrox ctpl phbC 225 195 0.02-6.28% lhydrox
ctpl bktB AVE: 1.0% lhydrox tpss phbB SD: 1.02% 36824 lhydrox ctpl
phbC 185 152 0.10-2.74% lhydrox ctpl bktB AVE: 0.6% lhydrox tpss
phbB SD: 0.5% lhydrox ctpl ilvA
[0123] The comparative results for PHA production in canola are
graphically presented in FIGS. 41 and 43. The beneficial results
obtained from the use of multigene vectors compared to results
obtained from traditional methods is visually impressive.
[0124] Since the promoters used in these two vectors sets (those
containing the 7s promoter and those containing the Lesquerella
hydroxylase promoter) are different, it cannot be distinguished
whether it was the Lesquerella promoter or the use of a single
vector that led to the increased polymer concentration. However, it
is clear that the single vector approach is viable for seed
expression of enzymes, including those required for PHA
biosynthesis. In addition, the increased speed of plant
construction and analysis using a single vector is a clear
benefit.
Example 8
Extraction of Polymer from Oilseed Rape and Analysis of Polymer
[0125] For isolation of polymer from canola seed, seeds were ground
to a fine powder with a mortar and pestle. Approximately 200 mg of
each sample were extracted two times with 10 mL each of hexane for
1 hour at 60.degree. C., then two times with 10 mL each of 100%
methanol for one hour at 60.degree. C. This procedure removed oil
from the seed. The material was allowed to dry completely
overnight. Polymer was extracted from the dried material with 1 mL
of chloroform containing 1 .mu.mol/mL methylbenzoate standard. The
tube was heated to 100.degree. C. for 5 hours, solid material was
removed by centrifugation, and the supernatant material was
subjected to methanolysis. Methanolysis of polymer and gas
chromatographic characterization of the methyl-ester residues were
performed as described by Slater et al. (J. Bacteriol. 180:
1979-1987, 1998).
Example 9
Multigene Vectors for Gene Expression in Mmonocots
[0126] For reasons described above, multigene vectors will also be
desirable for expression of multi-enzyme metabolic pathways in
monocots. Therefore, vectors designed to produce PHA in the leaves
of maize were constructed. These vectors use the e35s, eFMV, or
maize chlorophyll A/B binding protein (P-ChlA/B) promoters, and
include the HSP70 intron designed to enhance expression in
monocots. All enzymes were fused to the Arabidopsis RuBisCo small
subunit transit peptide. Other promoters might also be used.
Examples of vectors designed for gene expression in monocots are
pMON36843 (FIG. 26), pMON34543 (FIG. 27), and pMON36850 (FIG. 28).
These vectors have been used to transform maize, and polymer was
analyzed as described above for Arabidopsis. Polymer production is
summarized in Table 7.
7TABLE 7 Polymer production in maize using multigene vectors. Plant
construct # of plants # of plants C4 polymer Vector number
description assayed positive (% dry wt.) 36843 P-e35S phbC
1.14-4.81% P-e35S phbA 93 11 AVE: 1.84% P-e35S phbB SD: 1.04% 34543
P-eFMV phbC 34 34 0.15-2.95% P-eFMV phbA AVE: 0.7% P-eFMV phbB SD:
0.9% 36850 P-ChlA/B, phbC 132 78 0.1-5.66% P-ChlA/B, phbA AVE:
1.72% P-ChlA/B, phbB SD: 1.17%
Example 10
System for Construction of Large, Multigene Vectors
[0127] Since multigene vectors are optimal for producing high
levels of PHB, and this strategy is potentially optimal for
expression of other multiple step pathways, a simple method to
produce very large, multigene vectors is preferred. FIGS. 29 and 30
show plasmids pMON25963 and pMON25965, respectively. These vectors,
used together, provide a system for constructing very large
vectors. Plasmid pMON25965 provides a shuttle vector by which a
gene cassette can be cloned into the NotI restriction sites and
thereby be flanked by a series of restriction sites. These
restriction sites are relatively rare in many genomes, and thereby
of utility for subcloning many genes. Plasmid pMON25963 is a binary
vector designed for transformation of plants by Agrobacterium. It
contains a polylinker with the same sites found flanking the NotI
restriction sites of plasmid pMON25965. Using this system, a series
of gene "cassettes" can be produced using plasmid pMON25965, and
each can be sequentially ligated into plasmid pMON25963.
[0128] In practice, a series of vectors similar to pMON25965, but
having smaller polylinkers, will be preferred. Specifically, this
series of vectors would have a single NotI (or similar enzyme)
restriction site flanked by one or several other restriction enzyme
sites. By ligating cassettes flanked by large portions of the
pMON25965 polylinker into pMON25963, relatively large inverted
repeats of polylinker DNA are formed. These inverted repeats are
unstable in Escherichia coli, and plasmids harboring them do not
replicate efficiently. Thus, diminishing the size of the polylinker
in the shuttle vector can increase the probability of recovering
stable recombinants.
[0129] Another strategy for generating multigene vectors and
reducing the levels of background caused by vector re-ligation is
shown in FIG. 31. This strategy could be adapted to accommodate any
number of enzymes, depending on the availability of unique
restriction sites. One can easily design such a polylinker to
accommodate one's cloning needs. As the vector becomes larger, one
will want to have a larger homologous overlap for the ligation
process or choose restriction endonucleases producing ends that are
very easily ligated, and not self-compatible. By following the
cloning procedure outlined in FIG. 31, one can also control the
directionality of the clone. If directionality is not important
than clones generated from the ligation into the "shuttle vector"
in either orientation could be used. (A.rarw.C or A.fwdarw.C).
[0130] As with any multigene vector strategy, the starting plasmid
used for constructing the large multigene plasmids should be taken
into consideration. The common plant transformation plasmid pBIN19
(Frisch, D. et al., Plant Mol Biol 27: 405-409, 1995) has a
starting size of 11,777 bp. In contrast plasmid pMON10098 (FIG. 4)
has a starting size of 8431 bp. The major difference between the
two plasmids is the loss of the trfA function which is encoded in
trans in Agrobacterium strain ABI. Providing the trfA function in
trans allows replication only in the specific strains of
Agrobacterium engineered to harbor trfA. It has been shown by
Figurski and Helinski (Proc. Natl. Acad. Sci. U.S.A. 76: 1648-1652,
1979) that replication factors can function in trans. By providing
the minimal origins of replication required for maintenance in both
Escherichia coli and Agrobacterium the starting size of the initial
plasmid can be reduced significantly.
[0131] Other possibilities to reduce the size of the starting
plasmid would be to delete oriT since this sequence is required for
conjugational transfer only. If electroporation is used to
introduce the plasmid into Agrobacterium, oriT is not an essential
element. Another possibility would be to use selection that is
functional in plants, Agrobacterium, and Escherichia coli. This
could be accomplished by embedding into the plant promoter for the
selectable marker a suitable bacterial promoter sequence and a
ribosome binding site in proper context with the start codon on the
selectable marker. One could also place this selectable marker on
the plasmid flanked by its own right and left border sequences.
This may allow for the selectable marker to be integrated into the
plant chromosome unlinked to the genes of interest and potentially
removed from subsequent generations. Alternatively, plants could be
co-transformed by taking the multigene plasmid and cotransforming
on a separate plasmid the selectable marker for plants. This would
eliminate the cloning of the selectable marker on the multi gene
plasmid. The selectable marker can be delivered by mixing two
different Agrobacterium strains, one containing the multigene
plasmid and the other containing the selectable marker, or by using
the same Agrobacterium strain but having different isolates
containing either the multi gene plasmid or the selectable marker,
or by having the selectable marker coexisting in the same
Agrobacterium cell with the multigene vector, but on a separate
plasmid with a compatible origin of replication.
[0132] One can also envision reducing the size of the selectable
marker being used by using a trans complementation strategy. For
example, one could transform a plant with a portion of a NptII gene
that expresses a partial protein. If the transformation plasmid
carries the complementary portion of the NptII protein, both
fragments of the NptII protein may interact to confer resistance to
kanamycin. This is analogous to the .alpha.-complementation
strategy used for creating functional .beta.-galactosidase
(reviewed by Zabin, I. Mol. Cell. Biochem. 49: 87-96, 1982).
[0133] An example of an optimal starting plasmid for engineering
multiple genes in plants would contain only the minimal essential
elements required for replication in Escherichia coli and in
Agrobacterium (having all other required functions encoded in
trans) as well as a selection scheme that (1) reduces the need for
redundancy in the selectable marker, and/or (2) reduces the size of
the selectable marker, or (3) removes the necessity of having the
plant selectable marker on the multi gene plasmid. The promoter
used for driving the gene of interest in the multi gene vector
should consist of the minimal essential elements required for
temporal and spatial expression. The termination and
polyadenylation signals should also contain only those sequences
required for essential function.
Example 11
Poly(.beta.-hydroxybutyrate) Production in Oil Seed Leukoplasts of
Brassica napus
[0134] Using plants as factories is attractive for the production
of biodegradable plastics since current fermentation technology
used for the commercial production of polyhydroxyalkanoates (PHA)
is prohibitively expensive. The simplest PHA,
poly-.beta.-hydroxybutyrate (PHB), has previously been produced in
leaves of Arabidopsis thaliana (Nawrath, C., et al., Proc. Natl.
Acad. Sci., U.S.A., 91: 12760-12764, 1994). Brassica napus oilseed,
however, may provide a better system for PHB production because
acetyl-CoA, the substrate required in the first step of PHB
biosynthesis, is prevalent during fatty acid biosynthesis. Three
enzymatic activities are needed to synthesize the PHB polymer: a
.beta.-ketothiolase, an acetoacetyl-CoA reductase and a PHB
synthase. Genes from the bacterium Ralstonia eutropha encoding
these enzymes were independently engineered behind the
seed-specific Lesquerella fendleri oleate- 12 hydroxylase promoter
in a modular fashion. The gene cassettes were sequentially
transferred into a single, multi-gene vector which was used to
transform Brassica napus. PHB accumulated in leukoplasts to levels
as high as 7.7% of seed dry weight. Electron microscopy analyses
indicate that leukoplasts from these plants are distorted, yet
intact, and appear to expand in response to polymer
accumulation.
[0135] Polyhydroxyalkanoates (PHAs) comprise a class of
biodegradable polymers which offer an environmentally-sustainable
alternative to petroleum based plastics (reviewed by Poirier, Y.,
et al., Biotechnology, 13: 142-150, 1995). The homopolymer
Poly(.beta.-hydroxybutyrate) (PHB), a particularly well studied
PHA, is normally synthesized by various species of bacteria under
conditions where nutrients become limited. PHB is stored in
granules which can later be mobilized to provide a carbon and
energy resource for the bacteria.
[0136] One of the best-studied pathways for PHB synthesis is
derived from the bacterium Ralstonia eutropha (Slater, S. C., et
al, J. Bacteriol., 170: 4431-4436, 1988; Schubert, P., et al., J.
Bact., 170: 5837-47, 1988; Peoples, O. P., and Sinskey, A. J., J.
Biol. Chem., 264: 15298-15303, 1989; Peoples, O. P., and Sinskey,
A. J., J. Biol. Chem., 264: 15293-15297, 1989). The pathway
requires three enzymes: a .beta.-ketothiolase, an acetoacetyl-CoA
reductase, and a PHB synthase (FIG. 32). R. eutropha uses least two
.beta.-ketothiolases, PhbA and BktB (Slater, S. C., et al., J.
Bact., 180: 1979-1987, 1998), and both of these enzymes were used
in this study. The acetoacetyl-CoA reductase and PHB synthase are
designated PhbB and PhbC, respectively (Peoples, O. P., and
Sinskey, A. J., J. Biol. Chem, 264: 15298-15303, 1989; Peoples, O.
P., and Sinskey, A. J., J. Biol. Chem., 264: 15293-15297,
1989).
[0137] R. eutropha is fermented commercially for PHA production,
but the process is not economically competitive with polymers
derived from petroleum. Therefore, novel commercial efforts to
produce PHAs focus on using plants as polymer factories. In this
respect, our laboratory is considering two model systems:
production in leaves and production in seeds. Since acetyl-CoA is a
central metabolite for both PHB and fatty acid biosynthesis, and
Brassica napus seeds are extremely efficient in oil production, the
Brassica seeds seem an optimal environment in which to produce PHB
(U.S. Pat. No. 5,502,273). Production of PHB in Arabidopsis
thaliana leaves has been achieved using R. eutropha enzymes
(Poirier, Y., et al., Science, 256: 520-523, 1992), and additional
work showed that polymer accumulation up to 14% of plant dry weight
was achieved when the PHB biosynthetic enzymes were targeted to the
plastid (Nawrath, C., et al., Proc. Nat. Acad. Sci., 91:
12760-12764, 1994).
[0138] The work presented here demonstrates polymer production in
the seeds of Brassica napus using a multi-gene vector approach. A
significant advantage to using these multi-gene vectors is that the
entire PHA pathway is introduced simultaneously, thereby obviating
the need for elaborate crossing strategies and eliminating the
problems associated with insertional effects at multiple loci.
Construction of these multi-gene vectors involved the generation of
modular cassettes, each harboring an individual gene. The cassettes
were then assembled into a single vector expressing the entire PHB
biosynthetic pathway (FIG. 33). Each cassette consisted of the
Lesquerella fendleri oleate-12 hydroxylase promoter (Broun, P., et
al., Plant J., 13: 201-210, 1998), a chloroplast transit peptide
fused to the open reading frame of interest (bktB, phbA, phbB, or
phbC), and the 3' termination region of the Pisum sativum rbcSE9
gene (Coruzzi, G., et al., EMBO J., 3: 1671-1679, 1984). The
Lesquerella promoter contains 2.2 kb of DNA upstream of the coding
region for the oleate-12 hydroxylase gene. This promoter was chosen
because it is expressed concurrently with the accumulation of
storage lipid (Broun, P., et al., Plant J., 13: 201-210, 1998).
[0139] Expression of the PHB pathway in B. napus was achieved using
Agrobacterium-mediated transformation, and glyphosate selection was
used to identify transgenic events (Fry, J., et al., Plant Cell
Rep., 6: 321-325, 1987). The T-DNA transferred into the plants from
these experiments exceeded 16 kilobases in size. The co-expression
rate of genes from the multi-gene vectors in Brassica seeds was
high, with 87% of the glyphosate resistant plants also producing
polymer. Polymer levels ranged from 0.02-7.7% for the transgenic
plants carrying pMON36814 (R. eutropha phbA, phbB, phbC) and
0.02-6.3% for those carrying pMON36816 (R. eutropha bktB, phbB and
phbC). The vast majority of plants producing polymer fall within
the 0-3.0% polymer range (Table 8) and all polymer-producing lines
generated viable seed.
8TABLE 8 Polymer results from canola multigene vector
transformations. Genetic # of plants # of plants C4 polymer Vector
elements assayed positive (% dry wt.) p-Lh, phbC 0.02%-7.68% 36814
p-Lh, phbA 208 180 Avg: 1.73% p-Lh, phbB SD: 1.45% p-Lh, phbC
0.02%-6.28% 36816 p-Lh, bktB 225 195 Avg: 1.00% p-Lh, phbB SD:
1.02%
[0140] The B. napus line displaying 7.7% polymer was further
analyzed by electron microscopy. Micrographs revealed that polymer
accumulated within the plastid (FIG. 34), and that essentially
every plastid contained polymer. Polymer production in the plastids
is seemingly well tolerated; the size of the plastid expands to
accommodate polymer production (compare FIGS. 34A and 34B). This
phenomenon is similar to the size changes observed when amyloplasts
accumulate starch, and suggests that plastids will change size to
accommodate accumulation of any granular product. Thus, the signal
initiating an increase in plastid volume is not specifically linked
to accumulation of normal metabolites; rather, the increase is
probably initiated simply by physical pressure applied to the
plastid membrane.
[0141] These results demonstrate that PHA accumulation is possible
in an oilseed system. Commercial oilseed PHA production will
require approximately twice the amount of PHA accumulation achieved
here. Moreover, commercial success will rely on the development of
an integrated processing system to extract PHA, oil, and meal from
the seeds. We believe that increases in PHA accumulation can be
obtained using alternative promoters that are stronger and
expressed for a longer duration during seed development. Other
concerns regarding the feasibility of PHA production in planta
largely revolve around the metabolic effects of PHA production in
oilseeds. Specifically, analysis of the effect of PHA production on
oil yield will be of particular interest, since both are derived
from acetyl-CoA and produced simultaneously. Any untoward effect of
PHA production on oil yield or seed quality will impact negatively
on the economic feasibility of using B. napus as a commercial
system.
[0142] Vector Construction and Plant Transformation
[0143] A single vector encoding the entire PHB biosynthetic pathway
was used for Agrobacterium-mediated transformation of Brassica.
This vector, pMON36814, encodes bktB, phbB, and phbC (FIG. 33).
Each gene of interest was fused to a chloroplast transit peptide
(ctp), so each protein is transported to the seed leukoplast. All
enzymes were fused to the Arabidopsis RuBisCo small subunit la
transit peptide that was previously used for PHB production
(Nawrath, C, et al., Proc. Nat. Acad. Sci., 91: 12760-12764, 1994)
except PhbB was fused to the transit peptide from pea RuBisCo small
subunit (Cashmore, A. R., eds. Kosuge, T., Meredith C. P.,
Hollaender, A., (Plenum, N.Y.), 29-38, 1983). Each gene is
controlled by the promoter from the fatty acid hydroxylase gene of
Lesquerella (P-Lh; Broun, P., et al., Plant J., 13: 201-210, 1998),
and terminated with the E9 3' region of the Pisum rbcSE9 gene
(Coruzzi, G., et al., EMBO J., 3: 1671-1679, 1984). P-Lh directs
expression of these genes within the developing seed. The selection
cassette for pMON36812 and 36814 consisted of the Figwort Mosaic
Virus promoter followed by the Petunia RuBisCo small subunit 1a
transit peptide, the Petunia EPSP synthase gene (CP4) and nopaline
synthase 3' termination/polyadenylation region (nos3').
[0144] Transformation of Brassica napus was done as described in
Fry, J. et al. (Plant Cell Rep., 6: 321-325, 1987) using glyphosate
for selection.
[0145] Polymer Analysis
[0146] For isolation of polymer from canola seed, seeds were ground
to a fine powder with a mortar and pestle. Approximately 200 mg of
each sample were extracted two times in a glass tube with 10 mL
each of hexane for 1 hour at 60.degree. C., then two times with 10
mL each of 100% methanol for one hour at 60.degree. C. This
procedure removes oil from the seed. The material was allowed to
dry completely overnight. Polymer was extracted from the dried
material with 1 mL of chloroform containing 3 .mu.mol/mL
methyl-benzoate standard. The tube was heated to 100.degree. C. for
5 hours and the samples were cooled. One mL methanol/sulphuric acid
(85:15, v/v) was added, and the mixture was heated to 100.degree.
C. for exactly 2.5 hours. The solution was cooled, extracted with
water and subjected to gas chromatography. Gas chromatographic
characterization of the methyl-ester residues was performed as
described by (Slater, S., et al., J. Bact., 180: 1979-1987, 1998)
except that the temperature gradient was performed as follows: the
initial temperature of 70.degree. C. was held for 6 minutes, then
the temperature was increased by 30.degree. C. per minute to
130.degree. C. Finally, the temperature was increased by 50.degree.
C. per minute to 300.degree. C. and held at 300.degree. C. for 5
minutes.
[0147] Electron Microscopy
[0148] Partial imbibition of Brassica seeds was achieved by the
slight abrasion of the seed coats, followed by placement for 2
hours onto filter paper moistened with distilled water. The
cotyledons of these seeds were then cut into 1 mm.sup.3 pieces and
fixed in 4% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH
7.2 for three hours, with the first 30 minutes under vacuum. The
tissue was post-fixed in 1% osmium tetroxide in the above buffer,
dehydrated in ethanol and propylene oxide and infiltrated with a
1:1 mixture of Spurr's: EMbed 812 resin. The resin was polymerized
at 60.degree. C. for 48 hours. The resulting blocks were sectioned
on an Leica Ultracut E microtome. Sections 80 nm thick were picked
up on formvar/carbon coated copper slot grids. The grids were
post-stained with uranyl acetate and lead citrate in an LKB
ultrastainer and examined with a JEOL 1200 transmission electron
microscope. (All reagents were obtained from Electron Microscopy
Sciences, Fort Washington, Pa.).
Example 12
Metabolic Engineering of Arabidopsis and Brassica for
Poly(.beta.-hydroxybulyrate-co-.beta.-hydroxyvalerate) Copolymer
Production
[0149] Poly(hydroxyalkanoates) are natural polymers with
thermoplastic properties. One polymer of this class,
poly(.beta.-hydroxybutyrate-co-.be- ta.-hydroxyvalerate) (PHBV) is
currently produced by bacterial fermentation, but the process is
not economically competitive with polymer production from
petrochemicals. PHA production in green plants promises much lower
costs, but producing polymer with the appropriate monomer
composition is problematic. By redirecting metabolic pools of both
short-chain fatty acids and amino acids, Arabidopsis and Brassica
have now been engineered to produce PHBV, a copolymer with
commercial applicability. In this Example, polymer production,
metabolic intermediate analyses, and pathway dynamics for PHBV
synthesis in planta are described.
[0150] Poly(hydroxyalkanoates) (PHAs) are a class of polymers
accumulated by numerous bacterial species as carbon and energy
reserves. These polymers have thermoplastic properties, and have
received much attention as biodegradable alternatives to
petrochemical plastics (Anderson, A. J., and Dawes, E. A.
Microbiol. Rev. 54: 450-472, 1990). While the homopolymer
poly(.beta.-hydroxybutyrate) (PHB) is somewhat brittle, many
copolymers such as
poly(.beta.-hydroxybutyrate-co-.beta.-hydroxyvalerate) (PHBV) are
more flexible due to reduced crystallinity, and suitable for many
commercial applications.
[0151] The biochemical pathways for PHB and PHBV production are
essentially identical, differing only in the initial metabolites.
PHB synthesis is initiated by condensation of two acetyl-CoA
molecules, whereas PHBV synthesis requires the additional
condensation of acetyl-CoA with propionyl-CoA. Following
condensation, the products are reduced by a D-isomer specific
acetoacetyl-CoA reductase, and the resulting .beta.-hydroxy
products are polymerized by PHB synthase (Anderson, A. J., and
Dawes, E. A. Microbiol. Rev. 54: 450-472, 1990; Steinbuchel and
Schlegel, Mol. Microbiol. 5(3):535-42, 1991).
[0152] PHBV is produced commercially by growing Ralstonia eutropha
on glucose and propionate (Byrum, D. FEMS Microbiol. Rev. 102:
247-250, 1992), but the cost of this process prohibits large-scale
fermentation. Production of PHAs via genetic engineering of green
plants is expected to reduce costs to economical levels (van der
Leij, F. R., and Witholt, B. Can. J. Microbiol. 41(Suppl.1):
222-238, 1995), and production of PHB homopolymer in plants has
been demonstrated (Poirier, Y., et al. Science 256: 520-523, 1992;
Nawrath, C.; et al. Proc. Natl. Acad Sci. 91: 12760-12764, 1994).
However copolymer production has been problematic, primarily due to
the requirement for metabolic precursors other than acetyl-CoA.
[0153] Here we report metabolic engineering of plants to produce
PHBV copolymer. By expressing four distinct transgenes and
diverting metabolic pools of acetyl-CoA and threonine, copolymer
was produced in Arabidopsis thaliana, and in the seeds of Brassica
napus (oilseed rape). PHBV copolymer production opens the use of
green plants as factories for commercial,
environmentally-sustainable production of biodegradable
plastics.
[0154] Results: A Pathway for
Poly(.beta.-hydroxybutyrate-co-.beta.-hydrox- yvalerate) Production
in Plants
[0155] A pathway designed to engineer PHBV production in the
plastids of plants is diagrammed in FIG. 35. Acetyl-CoA is drawn
from plastid intermediary metabolism, whereas propionyl-CoA is
generated from threonine via 2-ketobutyrate (Gruys et al WO
98/00557; Eschenlauer, A. C., et al. Int. J. Biol. Macromol. 19:
121-130, 1996). This pathway requires transformation of the plant
with four separate genes: ilvA, bktB, phbB, and phbC. It also
relies on the endogenous plastid pyruvate dehydrogenase complex
(PDC). The threonine deaminase used in these studies is the
biosynthetic enzyme IlvA from E. coli (Taillon, B. E., et al. Gene
63: 245-252, 1988). The acetoacetyl-CoA reductase (PhbB) and PHB
synthase (PhbC) are the same R. eutropha enzymes used in earlier in
planta studies (Poirier, Y., et al. Science 256: 520-523, 1992;
Nawrath, C.; et al. Proc. Natl. Acad. Sci. 91: 12760-12764, 1994).
The .beta.-ketothiolase is BktB from R. eutropha (Slater, S., et
al. J. Bacteriol. 180: 1979-1987, 1998). Previous work on PHB
production in plants used the R. eutropha PhbA .beta.-ketothiolase.
However, PhbA cannot efficiently synthesize .beta.-ketovaleryl-CoA,
whereas BktB produces both .beta.-ketovaleryl-CoA and
acetoacetyl-CoA.
[0156] Metabolic Engineering of Arabidopsis and Brassica
[0157] Polymer production was studied in both Arabidopsis thaliana
leaves and Brassica napus seeds. For PHBV production in
Arabidopsis, two separate vectors were constructed. Plasmid
pMON25678 encodes phbB and phbC, and plasmid pMON25812 encodes bktB
and ilvA. Transgenic Arabidopsis were generated by simultaneous
Agrobacterium-mediated transformation with both vectors, and
subsequent selection on both glyphosate and kanamycin. All genes
were controlled by the e35S promoter (Odell, J. T., et al. Nature
313: 810-812, 1985), leading to polymer production throughout the
plant. In Brassica, all four genes in the transgenic pathway were
expressed from a single vector, pMON36824, and polymer production
was directed to the seeds by the Lesquerella hydroxylase promoter
(Broun, P., et al. Plant J. 13: 201-210, 1998).
[0158] Previous work on PHA production in plants has shown that
polymer is produced efficiently and that phenotypic effects on the
plant are minimized when PHA production occurs in the chloroplasts
(Nawrath, C. et al. Proc. Natl. Acad. Sci. 91: 12760-12764, 1994).
The plastids are the site for synthesis of both oil, which is
derived from acetyl-CoA, and threonine which is used to produce
propionyl-CoA. In both Arabidopsis and Brassica, the PHA
biosynthesis enzymes were targeted to the plastids using
chloroplast transit peptides. In photosynthetic tissues of
Arabidopsis the proteins are targeted to the chloroplasts, whereas
in Brassica seeds the enzymes are targeted to the leucoplasts.
[0159] Generation of Propionyl-CoA from Threonine
[0160] Conversion of threonine to 2-ketobutyrate by IlvA is the
first reaction catalyzed by one of the recombinantly-encoded
enzymes. IlvA normally catalyzes the initial step in the conversion
of threonine to isoleucine, and the enzyme is feedback-inhibited by
isoleucine (Umbarger, H. E. Biosynthesis of branched-chain amino
acids, pp. 442-457 in Escherichia coli and Salmonella: Cellular and
Molecular Biology, Neidhart, F. C., Curtiss, R., Lin, E. C. C.,
Low, K. B., Magasanik, B., Reznikoff, W. S., Riley, M., Schaechter,
M., and Umbarger, H. E. (eds.).ASM Press, Washington, D.C., 1996).
However, ilvA mutants with diminished sensitivity to isoleucine
have been described and two such mutants, ilvA466 (Pledger, W. J.,
and Umbarger, H. E. J. Bacteriol. 114: 183-194, 1973; Taillon, B.
E., et al. Gene 63: 245-252, 1988) and ilvA219 (Burns, R. O., et
al. J. Biol. Chem. 254: 1074-1079, 1979; Eisenstein, E., et al.
Biochemistry 34: 9403-9412, 1995), were used along with wild-type
ilvA in these studies. IlvA466 is partially sensitive to feedback
inhibition by isoleucine, and IlvA219 is essentially insensitive
(Pledger, W. J., and Umbarger, H. E. J. Bacteriol. 114: 195-207,
1973; LaRossa, R. A., et al. J. Bacteriol. 169: 1372-1378,
1987).
[0161] Both Arabidopsis and Brassica were initially transformed
with separate vectors expressing wild-type ilvA, ilvA466, and
ilvA219. In both organisms, no fertile transformants expressing
ilvA219 were recovered, indicating that expression of completely
isoleucine-insensitive IlvA is lethal. In Arabidopsis, plants
expressing ilvA466 were recovered at a very low frequency, whereas
Brassica tolerated ilvA466 rather well. This result may be due to
the seed-specific nature of the Lesquerella promoter. Transformants
expressing wild-type ilvA were efficiently recovered in both
Arabidopsis and Brassica.
[0162] In order to monitor the metabolic effects of IlvA in
transgenic plants, metabolites likely to be effected by this enzyme
were analyzed. FIG. 36 shows profiles of selected 2-ketoacids and
amino acids in a control plant, and in transgenic Arabidopsis
expressing wild-type ilvA. As expected, the transgenic plant had
elevated levels of both 2-ketobutyrate and isoleucine. In addition,
a high concentration of 2-aminobutyrate was present. Formation of
2-aminobutyrate from 2-ketobutyrate is a freely-reversible
reaction, probably catalyzed by the same branched-chain amino acid
transaminase that catalyzes the final step in isoleucine
biosynthesis (Singh, B. K. (1999) Biosynthesis of Valine, Leucine
and Isoleucine. In: Singh, B. K. (ed.) Plant Amino Acids:
Biochemistry and Biotechnology. Marcel Dekker, Inc., New York,
pp.227-247, 1998). Although transgenic plants expressing ilvA
contained more 2-ketobutyrate than did wild-type plants, the
2-ketobutyrate concentration was still below that of pyruvate. Most
2-ketobutyrate was apparently diverted to produce 2-aminobutyrate
and isoleucine. The concentration of free threonine in a plant
expressing ilvA decreased by only about 15%, suggesting that
threonine synthesis was sufficiently robust to compensate for the
diversion of threonine through 2-ketobutyrate. Similar analyses
were performed on the seeds from control and transgenic Brassica,
and essentially the same results were obtained. In plants
expressing ilvA, isoleucine, 2-ketobutyrate, and 2-aminobutyrate
concentrations were elevated, and free threonine was only
marginally decreased (K. Gruys et al., unpublished data).
[0163] The second step in the formation of propionyl-CoA is
catalyzed by the plastid pyruvate dehydrogenase complex, which is
the sole endogenous enzyme required for PHBV production. This
enzyme complex normally plays a central role in metabolism by
converting pyruvate to acetyl-CoA. We found that PDC from isolated
Brassica leukoplasts was also capable of converting 2-ketobutyrate
to propionyl-CoA. However, PDC was approximately 10-fold less
efficient when utilizing 2-ketobutyrate than when utilizing
pyruvate; the specific activities were 0.4 units/mg and 3.6
units/mg for 2-ketobutyrate and pyruvate, respectively.
[0164] Synthesis of PHBV Copolymer
[0165] Once propionyl-CoA has been produced, the pathway is
identical to that shown to produce PHBV copolymer in recombinant E.
Coli (Slater, S., et al. J. Bacteriol. 180: 1979-1987, 1998).
Propionyl-CoA is converted to D-.beta.-hydroxyvaleryl-CoA by BktB
and PhbB, and then is polymerized with D-.beta.-hydroxybutyryl-CoA
to form PHBV copolymer. The functionality of the entire pathway in
plants is shown in FIG. 37, which shows .sup.1H-NMR spectra
demonstrating the presence of PHBV copolymer in Arabidopsis. We
also obtained .sup.13C-NMR demonstrating PHBV copolymer production
in Brassica, and all these data have been corroborated by coupled
gas chromatography-mass spectrometry (data not shown). The
molecular weight of PHBV isolated from Brassica seeds was
approximately 1.times.10.sup.6, with a polydispersity index of 2.4.
These parameters are suitable for commercial applications.
[0166] Although copolymer was made in both Arabidopsis and
Brassica, the 3-hydroxyvalerate component varied with the in vivo
polymer concentration. The polymer composition in Brassica seeds
distinctly showed a negative correlation between the
3-hydroxyvalerate content of the polymer and total polymer
production (FIG. 38). Threonine deaminase activity also negatively
correlated with 3-HV content (FIG. 38), a somewhat surprising
result considering the role of IlvA in the production of 3-HV.
However, we have consistently found that introduction of vectors
encoding multiple genes leads to a general, concerted expression of
all encoded enzymes. Thus, elevated IlvA activity is consistent
with elevated polymer production.
[0167] Discussion
[0168] The use of green plants as industrial factories will often
require significant changes in plant metabolism, so metabolic
engineering of multi-step pathways will become an important
technology in "green chemistry" efforts. In this study, production
of the PHA copolymer PHBV has been accomplished using a combination
of endogenous and transgene-encoded enzymes. The pathway consists
of five separate enzymes, four being encoded as transgenes. In the
case of Brassica, all four genes were successfully introduced on a
single vector.
[0169] Commercial application of this technology will rest on two
primary metabolic issues: 1) can polymer be produced in planta to
concentrations amenable to economical polymer extraction? and 2) as
the polymer concentration increases, can the appropriate monomer
composition be maintained? We expect that polymer concentrations in
planta will need to reach at least 15% of dry weight for economical
production to be feasible. PHB homopolymer concentrations near 15%
have been reported (Nawrath, C. et al. Proc. Natl. Acad. Sci. 91:
12760-12764, 1994) and have also been achieved in our laboratory
(data not shown). Thus, high-level PHB production appears
technically attainable.
[0170] Production of PHBV copolymer has been accomplished in this
study, although all plants produced copolymer at levels below 3% of
plant tissue dry weight. The next challenge is high-level
production of copolymer, and the data in FIG. 38 show that
additional work is required to maintain the 3-hydroxyvalerate
composition at high polymer concentrations. Specifically, as
polymer production increased, the 3-hydroxyvalerate fraction of the
polymer decreased, and increasing threonine deaminase expression
did not effect this correlation. These data suggest a metabolic
bottleneck in the provision of 3-hydroxyvalerate to PHA synthase.
The BktB, PhbB, PhbC pathway efficiently synthesizes PHBV copolymer
(Slater, S., et al. J. Bacteriol. 180: 1979-1987, 1998), and
production of 2-ketobutyrate in planta is efficient, as estimated
from the elevated levels of 2-ketobutyrate, 2-aminobutyrate and
isoleucine (FIG. 36). Thus, the metabolic bottleneck must exist at
the conversion of 2-ketobutyrate to propionyl-CoA by the pyruvate
dehydrogenase complex. As noted above, the PDC strongly prefers
pyruvate as a substrate, and this difference is compounded in vivo
by the concentration ratio of pyruvate to 2-ketobutyrate (FIG. 36).
Pyruvate dehydrogenase apparently cannot effectively compete for
2-ketobutyrate so propionyl-CoA synthesis is limited..
[0171] Production of copolymer to high internal concentrations may
require a supplementary route for conversion of 2-ketobutyrate to
propionyl-CoA. There are several ways to bypass the PDC or
supplement its activity, but all will require additional
transgenes. These routes include modifying the .alpha.-ketoacid
dehydrogenase to more readily accept propionyl-CoA (Inoue H, et al.
J. Bacteriol. 179: 3956-3962, 1997; Gruys et al WO 98/00557),
expression of an alternative enzyme complex capable of forming
propionyl-CoA from 2-ketobutyrate (Kerscher, L. and Oesterhelt, D.,
Eur. J. Biochem. 116: 587-594, 1981), or co-expression of a
propionyl-CoA dehydrogenase (Horswill et al; Mitsky et al.,
unpublished data) with a propionyl-CoA synthetase or CoA
transferase (Gruys et al WO 98/00557; Valentin et al, manuscript in
preparation). Thus, a commercially viable transgenic plant
producing PHA polymer from threonine may contain up to six separate
transgenes.
[0172] Synthesis of propionyl-CoA can also be achieved through
other metabolic pathways, although none presents a straightforward
alternative to the threonine derived pathway (FIG. 39). For
instance, propionyl-CoA may be generated from acetyl-CoA using a
5-step pathway, part of which is involved in propionyl-CoA
degradation in plants (Goodwin, T. W. and Mercer, E. I.
Introduction to Plant Biochemistry. Second Edition. Pergamon Press,
Oxford, 1985; Eisenreich, W., et al. Eur. J. Biochem. 215: 619-632,
1993; Preifert, H., and Steinbuchel, A. J. Bacteriol. 174:
6590-6599, 1992; Podkowinski, J., et al. Proc. Natl. Acad. Sci. USA
93: 1870-1874, 1996; Sun, J., et al. Plant Physiol. 115: 1371-1383,
1997; Horswill A. R., and Escalante-Semerena J. C. J. Bacteriol.
179: 928-940, 1997; Gruys et al, unpublished data). Conversion of
acrylyl-CoA to propionyl-CoA is potentially problematic, but an
appropriate enzyme may be available from Chroroflexus aurantiacus
(Eisenreich, W., et al. Eur. J. Biochem. 215: 619-632, 1993).
Propionyl-CoA can also be derived from succinyl-CoA using a pathway
present in both Rhodococcus ruber and Nocardia corallina (Williams,
D. R.,et al. Appl. Microbiol. Biotechnol. 40: 717-723, 1994;
Valentin, H. E., and Dennis, D. Appl. Environ. Microbiol. 62:
372-379, 1996). This pathway is initiated by methylmalonyl-CoA
mutase, an enzyme that requires vitamin B.sub.12 as a cofactor.
However, vitamin B.sub.12 is not synthesized in plants (Goodwin, T.
W. and Mercer, E. I. Introduction to Plant Biochemistry. Second
Edition. Pergamon Press, Oxford, 1985). Rhodococcus and Nocardia
also produce minor amounts of 3-hydroxyvaleryl-CoA via a different,
uncharacterized route. This route may be a link to amino acid
metabolism, such as the pathways used by other bacteria and animals
to degrade valine and isoleucine (FIG. 39). These pathways might
also be engineered in plants, but a large number of genes are
required.
[0173] Several other amino acids can be used to produce
propionyl-CoA. Methionine, like threonine, generates 2-ketobutyrate
during catabolism. This conversion is catalyzed by L-methionine
.gamma.-lyase in a reaction that also produces ammonia and
methanethiol (Tanaka, H., et al. Enzyme Microb. Technol. 7:
530-537, 1985). The effect of methanethiol production on plants is
unknown, and supplementation of PDC activity would still be
required to efficiently produce propionyl-CoA. Another pathway,
present in Clostridium propionicum, converts alanine to
propionyl-CoA via lactic acid, lactyl-CoA and acrylyl-CoA
(Schweiger, G., and Buckel, W. FEBS Lett. 171: 79-84, 1984; Cardon,
B. P., and Barker, H. A. Arch. Biochem. Biophys. 12: 165-180,
1947). However, none of the required genes has been cloned, and
some of the necessary enzymes are oxygen sensitive (Hofmeister, A.
E. M., and Buckel, W. Eur. J. Biochem. 206: 547-552, 1992; Kuchta,
R. D., and Abeles, R. H. J. Biol. Chem. 260: 13181-13189, 1985).
.beta.-alanine is another potential starting metabolite for the
production of propionyl-CoA (Arst, H. N. Jr. Mol. Gen. Genet. 163:
23-27, 1978; Roberts, E., and Bregoff, H. M. J. Biol. Chem. 201:
393-398, 1953; Kupiecki, R. P., and Coon, M. J. J. Biol. Chem. 229:
743-754, 1957). .beta.-alanine normally plays a critical role as a
precursor to Coenzyme-A and acyl carrier protein. However, little
is known about the concentration and compartmentalization of
.beta.-alanine in plants, and propionyl-CoA may actually be
required for its synthesis.
[0174] In summary,
poly(.beta.-hydroxybutyrate-co-.beta.-hydroxyvalerate) copolymer
was produced in both Arabidopsis and Brassica by simultaneously
accessing amino acid and short-chain fatty acid metabolite pools.
In Brassica, all four required transgenes were introduced on a
single vector, eliminating the plant crossing normally necessary to
assemble a pathway of this size. The polymer molecular mass was
adequate for commercial purposes, but an apparent metabolic
bottleneck in conversion of 2-ketobutyrate to propionyl-CoA
suggests that additional engineering may be required to achieve
high-level production of polymer with the necessary
P-hydroxyvalerate composition.
[0175] Generation of IlvA Mutants
[0176] All ilvA alleles used herein are derived from the E. coli
ilvA gene (Lawther, R. P. et al., Nucl. Acids Res. 11: 2137-2155,
1987) that is harbored in pMON25659 (Gruys et al WO 98/00557). The
ilvA219 mutation (Eisenstein, E., et al. Biochemistry. 34:
9403-9412, 1995) and ilvA466 mutation (Taillon, B. E., et al. Gene.
63: 245-252, 1988), both originally isolated in Salmonella
typhimurium, were introduced into the E. coli gene by
oligonucleotide-directed mutagenesis as previously described (Gruys
et al. WO 98/00557).
[0177] Plasmid Construction and Transformation of Arabidopsis
Thaliana and Brassica Napus
[0178] All transformation vectors are derived from pMON10098, a
vector designed for Agrobacterium-mediated transformation of plants
that encodes the nptII selectable marker. The trfA function is
provided in trans by the host bacterium, Agrobacterium tumefaciens
ABI. A. tumefaciens ABI is Agrobacterium strain GV3101 (Van
Larebeke, N., et al. Nature. 252: 169-170, 1974) harboring the
helper plasmid pMP9ORK (Koncz, C., and Schell, J. Mol. Gen. Genet.
204: 383-396, 1986).
[0179] All PHA production genes used in this study were initially
constructed in intermediate vectors as cassettes including a
promoter, a chloroplast transit peptide fused to the gene of
interest, and a 3' control region. In every case, the gene cassette
is flanked by Not I restriction sites, plus several additional
unique restriction sites. Each cassette was excised from it's
intermediate vector using appropriate restriction enzymes, and
sequentially ligated into the recombinant vector for plant
transformation.
[0180] For metabolite analysis, Arabidopsis was transformed with
either pMON15715, an ilvA-negative control vector, or pMON25668,
which expresses both phbA and wild-type ilvA from e35S
promoters.
[0181] For production of PHBV in Arabidopsis, two separate plasmids
were used.
[0182] The first vector encoded both phbB and phbC (pMON25678), and
the second vector encoded both bktB and ilvA (pMON25812). All genes
were controlled by the e35S promoter (Odell, J. T., et al. Nature.
313: 810-812, 1995) and the E9 3' region (Coruzzi, G., et al. EMBO
J. 3: 1671-1679, 1984). All enzymes were fused to the Arabidopsis
RuBisCo small subunit la transit peptide that was previously used
for PHB production (Nawrath, C., et al. Proc. Natl. Acad. Sci. 91:
12760-12764, 1994). Plasmid pMON25678 encodes resistance to
glyphosate, whereas pMON25812 encodes resistance to kanamycin. Both
plasmids were simultaneously used for Agrobacterium-mediated
Arabidopsis transformation (Bechtold N., et al. Comptes Rendus
Acad. Sci. Paris Sciences Serie III Sciences de la Vie. 316:
1194-1199, 1993), and transformants were selected on both
glyphosate and kanamycin as follows.
[0183] Arabidopsis thaliana Columbia plants were grown in Metro Mix
200 in 2.5 in. pots covered with a mesh screen. Sown seed was
vernalized for 5 days and germinated under conditions of 16 hours
light/8 hours dark at 20.degree. C. to 22.degree. C., 75% humidity.
Plants were watered and fertilized twice weekly with 1/2X Peters
20-20-20 until infiltration.
[0184] A 1:50 dilution of an overnight culture of Agrobacterium
tumefaciens ABI strain was grown at 28.degree. C. in YEP containing
Spectinomycin 100 mg/L, Streptomycin, 100 mg/L, Chloramphenicol 25
mg/L, and Kanamycin 50 mg/L. Each culture contained a different ABI
construct. After 16-20 hours the Agrobacterium cultures were
concentrated by centrifugation. The supernatant was discarded and
the cell pellets were dried and resuspended in infiltration medium
(MS Basal Salts 0.5%, Gamborg's B-5 Vitamins 1%, Sucrose 5%, MES
0.5 g/L, pH 5.7) with 0.44 nM benzylaminopurine (10 .mu.L of a 1.0
mg/L stock in DMSO per liter) and 0.02% Silwet L-77 to an
OD.sub.600 of 0.8. For co-infiltrations each culture was
resuspended as described above and 150 mL each of two cultures were
combined for a total of 300 mL.
[0185] Plants were soaked in water 30 minutes prior to
infiltration. Inverted plants were placed into the cultures and
vacuum infiltrated at 27 in. Hg for 10 minutes. The plants were
placed on their sides in a diaper-lined tray and covered with a
germination dome for one day. The pots were then turned upright and
were not watered for five days. Infiltrated plants were grown to
maturity as described above. Ripe seeds were harvested and
sterilized. Harvested seed was placed in a 15 mL Corning tube and
sterilized. The tubes containing seed were placed on their sides
with lids loosened in a vacuum dessicator containing a beaker of
Clorox and 1:100 hydrochloric acid. The dessicator was then sealed
with a vacuum and the seed remained in the dessicator overnight.
Sterilized seeds from co-infiltrated plants were placed on media
containing MS Basal Salts 4.3 g/L, Gamborg's B-5 (500.times.) 2.0
g/L, glucose 10 g/L, MES 0.5 g/L, and 8 g/L phytagar with
carbenicillin 250 mg/L, cefotaxime 100 mg/L, kanamycin 60 mg/L and
4 mM glyphosate. The seed was germinated at 26.degree. C., 20 hours
light/4 hours dark. Transformants were transferred to soil and
covered with a germination dome for one week. The plants were grown
in plant growth conditions described above.
[0186] For transformation of Brassica napus, a single vector
encoding the entire PHBV biosynthesis pathway was used. This
vector, pMON36824, encodes bktB, phbB, phbC, and ilvA466 (FIG. 3).
As with the Arabidopsis vectors, each gene of interest was fused to
a chloroplast transit peptide, so each protein is transported to
the seed leukoplast. All enzymes were fused to the Arabidopsis
RuBisCo small subunit 1 a transit peptide that was previously used
for PHB production (Nawrath, C. et al. Proc. Natl. Acad. Sci. 91:
12760-12764, 1994), except PhbB was fused to the transit peptide
from pea RuBisCo small subunit (Cashmore, A. R. Nuclear genes
encoding the small subunit of ribulose-1,5-bisphosphate
carboxylase. pp. 29-38 in Genetic Engineering of Plants, Kosuge,
T., Meredith, C. P., Hollaender, A. (eds.). Plenum, N.Y., 1983).
Each gene is controlled by the promoter from the fatty acid
hydroxylase gene of Lesquerella (P-Lh; Broun, P., et al. Plant J.
13: 201-210, 1998), and the E9 3' region (Coruzzi, G., et al. EMBO
J. 3: 1671-1679, 1984). P-Lh directs expression of these genes
within the developing seed. Transformation of Brassica was
performed as described by Fry et al. (Plant Cell Rep. 6: 321-325,
1987), and transformants were selected on glyphosate.
[0187] Isolation of Brassica Seed Leukoplasts and Analysis of
Pyruvate Dehydrogenase Complex Activity
[0188] Leukoplasts were isolated essentially as described by Kang
and Rawsthome (Plant J. 6: 795-805, 1994). Isolated leucoplasts
were lysed by sonication and debris removed by centrifugation at
10,000.times. g for 10 minutes. The crude extract was desalted
using Pharmacia NAP-5 columns and the protein concentrations
determined by the Bradford method (Bradford, M. Anal. Biochem. 72:
248-254, 1976). Five to 50 .mu.L were added to assay mix which
contained final concentrations of: 100 mM EPPS, pH 8.0; 5 mM
MgCl.sub.2; 2.4 mM coenzyme-A; 1.5 mM NAD.sup.+; and 0.2 mM TPP
(cocarboxylase). The reaction was initiated with addition of either
pyruvate or 2-ketobutyrate substrates to final concentrations of
1.5 mM and 30 mM, respectively. To aid in analysis and ensure peak
identities, .sup.14C labeled pyruvate and 2-ketobutyrate were
spiked into both substrates. The reactions were quenched with 30
.mu.L of 10% formic acid after 2 to 30 minutes. 100 .mu.L of the
reaction was injected onto a Beckman Ultrasphere HPLC column (5
.mu.M, 4.6 mm.times.15 cm) and eluted with 1 mL/minute gradient of
solvent A (50 mM ammonium acetate buffer pH 6.0 containing 5%
acetonitrile) going from 0 to 40 % solvent B (acetonitrile) in 15
minutes. The reaction was followed by monitoring absorbance of
CoA-derived products at 230 and 260 nm using a photodiode array
detector. Use of radioisotope flow detector allowed confirmation of
both substrate and product peak identities. The percent conversion
of added substrates was used to determine the specific activities
of the extracts. One unit equals one nmol product produced per
minute per mg protein in extract.
[0189] Amino Acid and 2-Ketoacid Analysis
[0190] Amino Acid analysis was performed by Dr. Donald Willis at
Ralston Analytical Laboratories, essentially as described by Willis
(J. Chromatog. 408: 217-225, 1987).
[0191] Extraction and Gas Chromatography Analysis of Polymer from
Arabidopsis
[0192] For isolation of polymer from Arabidopsis, stems and leaves
were harvested and dehydrated by lyophilization for approximately
36 hours. The material was ground to a fine powder, and 100 mg of
powder was treated with 10 mL Clorox bleach for 1 hour with shaking
at room temperature. The extract was subjected to centrifugation at
1,600.times. g for ten minutes, and the supernatant solutions was
carefully removed. Ten mL 100% methanol were added, the solution
was mixed by vortex, and then centrifuged again. After a second,
identical, methanol extraction, the material was allowed to dry
overnight. Polymer was extracted from the dried material with 1 mL
of chloroform containing 3 .mu.mol/mL methyl-benzoate standard and
1 mL of methanol/sulphuric acid (85:15, v/v). The tube was heated
to 100.degree. C. for exactly 2.5 hours, and the solid material was
removed by centrifugation. The solution was cooled, 1 mL water was
added, and the liquid was mixed using a vortex mixer. The organic
and aqueous phases were separated by centrifugation at 1,600.times.
g for ten minutes. The chloroform layer was transferred to a clean
test tube and vigorously mixed with approximately 200 mg of silica
gel. Solid material was removed by centrifugation, and the
supernatant material was subjected to gas chromatography. Gas
chromatographic characterization of the methyl-ester residues was
performed as described by Slater et al. (J. Bacteriol. 180:
1979-1987, 1998), except that the temperature gradient was
performed as follows. The initial temperature of 70.degree. C. was
held for 6 minutes, then the temperature was increased by
30.degree. C. per minute to 130.degree. C. Finally, the temperature
was increased by 50.degree. C. per minute to 300.degree. C. and
held at 300.degree. C. for 5 minutes.
[0193] Extraction and Gas Chromatography Analysis of Polymer from
Brassica seeds
[0194] For isolation of polymer from canola seed, seeds were ground
to a fine powder with a mortar and pestle. Approximately 200 mg of
each sample were extracted two times in a glass tube with 10 mL
each of hexane for 1 hour at 60.degree. C., then two times with 10
mL each of 100% methanol for one hour at 60.degree. C. This
procedure removes oil from the seed. The material was allowed to
dry to completion overnight. Polymer was extracted from the dried
material with 1 mL of chloroform containing 3 .mu.mol/mL
methyl-benzoate standard. The tube was heated to 100.degree. C. for
5 hours and the samples were cooled. One mL methanol/sulphuric acid
(85:15, v/v) was added, and the mixture was heated to 100.degree.
C. for exactly 2.5 hours. The solution was cooled, extracted with
water and subjected to gas chromatography as described above.
[0195] Characterization of Polymer by Nuclear Magnetic Resonance
Spectroscopy and Gel Permeation Chromatography
[0196] Nuclear magnetic resonance (NMR) studies were done using a
Varian Unity 500 MHz spectrometer. Proton spectra were obtained on
a Varian pfg 5 mm probe at 30.degree. C. from PHA samples of
approximately 20 mg dissolved in 1 mL deuterochloroform.
Acquisitions were taken at a 90.degree. pulse, 2.3 s acquisition
time, 30 s delay, collecting 65 k data points and 16 accumulations.
Chemical shifts were referenced to CHCl.sub.3 (.delta.=7.24 ppm).
The 13C{1H} spectra (125 MHz) were taken at 30.degree. C. on a
Nalorac 3 mm .sup.13C probe containing a solution of approximately
10 mg PHA in 200 .mu.L deuterochloroform. The spectra were obtained
using 30.degree. pulses, 1.5 s acquisition time, zero delay, 131 k
data points and 55,296 accumulations. Chemical shifts were measured
relative to CHCl.sub.3 (.delta.=77.0 ppm).
[0197] Gel permeation chromatography was performed according to
Koizumi et al. (J. M. S. Pure Appl. Chem. A32: 759-774,1995).
Example 13
Plant Promoters
[0198] Plant promoter sequences can be constitutive or inducible,
environmentally- or developmentally-regulated, or cell- or
tissue-specific. Often-used constitutive promoters include the CaMV
35S promoter (Odell et al., Nature 313: 810-812, 1985), the
enhanced CaMV 35S promoter, the Figwort Mosaic Virus (FMV) promoter
(Richins, R. D. et al., Nucleic Acids Res. 20: 8451-8466, 1987),
the mannopine synthase (mas) promoter, the nopaline synthase (nos)
promoter, and the octopine synthase (ocs) promoter. Useful
inducible promoters include promoters induced by salicylic acid or
polyacrylic acids (PR-1, Willians , S. W. et al, Biotechnology 10:
540-543, 1992), induced by application of safeners (substituted
benzenesulfonamide herbicides, Hershey, H. P. and Stoner, T. D.,
Plant Mol. Biol. 17: 679-690, 1991), heat-shock promoters (Ou-Lee
et al., Proc. Natl. Acad. Sci. U.S.A. 83: 6815-6819, 1986; Ainley,
W. M. et al., Plant Mol. Biol. 14: 949-967, 1990), a
nitrate-inducible promoter derived from the spinach nitrite
reductase gene (Back, E. et al., Plant Mol. Biol. 17: 9-18, 1991),
hormone-inducible promoters (Yamaguchi-Shinozaki, K. et al., Plant
Mol. Biol. 15: 905-912, 1990; Kares et al., Plant Mol. Biol. 15:
905-912, 1990), and light-inducible promoters associated with the
small subunit of RuBP carboxylase and LHCP gene families
(Kuhlemeier et al., Plant Cell 1: 471-478, 1989; Feinbaum, R. L. et
al., Mol. Gen. Genet. 226: 449-456, 1991; Weisshaar, B. et al.,
EMBO J. 10: 1777-1786, 1991; Lam, E. and Chua, N. H., J. BioL Chem.
266: 17131-17135, 1990; Castresana, C. et al., EMBO J. 7:
1929-1936, 1988; Schulze-Lefert, P. et al., EMBO J. 8: 651-656,
1989). Examples of useful tissue-specific,
developmentally-regulated promoters include the .beta.-conglycinin
7S promoter (Doyle, J. J. et al., J. Biol. Chem. 261: 9228-9238,
1986; Slighton and Beachy, Planta 172: 356, 1987), and
seed-specific promoters (Knutzon, D. S. et al., Proc. Natl. Acad.
Sci. U.S.A. 89: 2624-2628, 1992; Bustos, M. M. et al., EMBO J. 10:
1469-1479, 1991; Lam, E. and Chua, N. H., Science 248: 471-474,
1991; Stayton et al., Aust. J. Plant. Physiol. 18: 507, 1991).
Plant functional promoters useful for preferential expression in
seed plastids include those from plant storage protein genes and
from genes involved in fatty acid biosynthesis in oilseeds.
Examples of such promoters include the 5' regulatory regions from
such genes as napin (Kridl et al., Seed Sci. Res. 1: 209-219,
1991), phaseolin, zein, soybean trypsin inhibitor, ACP,
stearoyl-ACP desaturase, and oleosin. Seed-specific gene regulation
is discussed in EP 0 255 378. Promoter hybrids can also be
constructed to enhance transcriptional activity (Comai, L. and
Moran, P. M., U.S. Pat. No. 5,106,739, issued Apr. 21, 1992), or to
combine desired transcriptional activity and tissue
specificity.
Example 14
[0199] Plant Transformation and Regeneration
[0200] A variety of different methods can be employed to introduce
such vectors into plant protoplasts, cells, callus tissue, leaf
discs, meristems, etcetera, to generate transgenic plants,
including Agrobacterium-mediated transformation, particle gun
delivery, microinjection, electroporation, polyethylene glycol
mediated protoplast transformation, liposome-mediated
transformation, etc. (reviewed in Potrykus, Ann. Rev. Plant
Physiol. Plant Mol. Biol. 42: 205-225, 1991). In general,
transgenic plants comprising cells containing and expressing DNAs
encoding enzymes facilitating PHA biosynthesis can be produced by
transforming plant cells with a DNA construct as described above
via any of the foregoing methods; selecting plant cells that have
been transformed on a selective medium; regenerating plant cells
that have been transformed to produce differentiated plants; and
selecting a transformed plant which expresses the enzyme-encoding
nucleotide sequence.
[0201] Specific methods for transforming a wide variety of dicots
and obtaining transgenic plants are well documented in the
literature (Gasser and Fraley, Science 244: 1293-1299, 1989; Fisk
and Dandekar, Scientia Horticulturae 55: 5-36, 1993; Christou, Agro
Food Industry Hi Tech, p.17 (1994); and the references cited
therein).
[0202] Successful transformation and plant regeneration have been
reported in the monocots as follows: asparagus (Asparagus
officinalis; Bytebier et al., Proc. Natl. Acad. Sci. U.S.A. 84:
5345-5349, 1987); barley (Hordeum vulgarae; Wan and Lemaux, Plant
Physiol. 104: 37-48, 1994); maize (Zea mays; Rhodes, C. A. et al.,
Science 240: 204-207, 1988; Gordon-Kamm et al., Plant Cell 2:
603-618, 1990; Fromm, M. E. et al., Bio/Technology 8: 833-839,
1990; Koziel et al., Bio/Technology 11: 194-200, 1993); oats (Avena
sativa; Somers et al., Bio/Technology 10: 1589-1594, 1992);
orchardgrass (Dactylis glomerata; Horn et al., Plant Cell Rep. 7:
469-472, 1988); rice (Oryza sativa, including indica and japonica
varieties; Toriyama et al., Bio/Technology 6: 10, 1988; Zhang et
al., Plant Cell Rep. 7: 379-384, 1988; Luo and Wu, Plant Mol. Biol.
Rep. 6: 165, 1988; Zhang and Wu, Theor. Appl. Genet. 76: 835, 1988;
Christou et al., Bio/Technology 9: 957-962, 1991); rye (Secale
cereale; De la Pena et al., Nature 325: 274-276, 1987); sorghum
(Sorghum bicolor; Casas, A. M. et al., Proc. Natl. Acad. Sci.
U.S.A. 90: 11212-11216, 1993); sugar cane (Saccharum spp.; Bower
and Birch, Plant J. 2: 409-416, 1992); tall fescue (Festuca
arundinacea; Wang, Z. Y. et al., Bio/Technology 10: 691-696, 1992);
turfgrass (Agrostis palustris; Zhong et al., Plant Cell Rep. 13:
1-6, 1993); wheat (Triticum aestivum; Vasil et al., Bio/Technology
10: 667-674, 1992; Weeks, T. et al., Plant Physiol. 102: 1077-1084,
1993; Becker et al., Plant J. 5: 299-307, 1994), and alfalfa
(Masoud, S. A. et al., Transgen. Res. 5: 313, 1996).
Example 15
Host Plants
[0203] Particularly useful plants for polyhydroxyalkanoate
production include those that produce carbon substrates which can
be employed for polyhydroxyalkanoate biosynthesis, including
tobacco, wheat, potato, Arabidopsis, and high oil seed plants such
as corn, soybean, canola, oil seed rape, sunflower, flax, peanut,
sugarcane, switchgrass, and alfalfa.
[0204] If the host plant of choice does not produce the requisite
fatty acid substrates in sufficient quantities, it can be modified,
for example by mutagenesis or genetic transformation, to block or
modulate the glycerol ester and fatty acid biosynthesis or
degradation pathways so that it accumulates the appropriate
substrates for polyhydroxyalkanoate production. Expression of
enzymes such as acyl-ACP thioesterase, fatty acyl hydroxylase, and
yeast MFP may serve to increase the flux of substrates in the
peroxysome, leading to higher levels of polyhydroxyalkanoate
biosynthesis.
EXAMPLE 16
Nucleic Acid Mutation and Hybridization
[0205] Variations in the nucleic acid sequence encoding a fusion
protein may lead to mutant protein sequences that display
equivalent or superior enzymatic characteristics when compared to
the sequences disclosed herein. This invention accordingly
encompasses nucleic acid sequences which are similar to the
sequences disclosed herein, protein sequences which are similar to
the sequences disclosed herein, and the nucleic acid sequences that
encode them. Mutations may include deletions, insertions,
truncations, substitutions, fusions, and the like.
[0206] Mutations to a nucleic acid sequence may be introduced in
either a specific or random manner, both of which are well known to
those of skill in the art of molecular biology. A myriad of
site-directed mutagenesis techniques exist, typically using
oligonucleotides to introduce mutations at specific locations in a
nucleic acid sequence. Examples include single strand rescue
(Kunkel, T. Proc. Natl. Acad Sci. U.S.A., 82: 488-492, 1985),
unique site elimination (Deng and Nickloff, Anal. Biochem. 200: 81,
1992), nick protection (Vandeyar, et al. Gene 65: 129-133, 1988),
and PCR (Costa, et al. Methods Mol. Biol. 57: 31-44, 1996). Random
or non-specific mutations may be generated by chemical agents (for
a general review, see Singer and Kusmierek, Ann. Rev. Biochem. 52:
655-693, 1982) such as nitrosoguanidine (Cerda-Olmedo et al., J.
Mol. Biol. 33: 705-719, 1968; Guerola, et al. Nature New Biol. 230:
122-125, 1971) and 2-aminopurine (Rogan -and Bessman, J. Bacteriol.
103: 622-633, 1970), or by biological methods such as passage
through mutator strains (Greener et al. Mol. Biotechnol. 7:
189-195, 1997).
[0207] Nucleic acid hybridization is a technique well known to
those of skill in the art of DNA manipulation. The hybridization
properties of a given pair of nucleic acids is an indication of
their similarity or identity. Mutated nucleic acid sequences may be
selected for their similarity to the disclosed nucleic acid
sequences on the basis of their hybridization to the disclosed
sequences. Low stringency conditions may be used to select
sequences with multiple mutations. One may wish to employ
conditions such as about 0.15 M to about 0.9 M sodium chloride, at
temperatures ranging from about 20.degree. C. to about 55.degree.
C. High stringency conditions may be used to select for nucleic
acid sequences with higher degrees of identity to the disclosed
sequences. Conditions employed may include about 0.02 M to about
0.15 M sodium chloride, about 0.5% to about 5% casein, about 0.02%
SDS and/or about 0.1% N-laurylsarcosine, about 0.001 M to about
0.03 M sodium citrate, at temperatures between about 50.degree. C.
and about 70.degree. C. More preferably, high stringency conditions
are 0.02 M sodium chloride, 0.5% casein, 0.02% SDS, 0.001 M sodium
citrate, at a temperature of 50.degree. C.
Example 17
Determination of Homologous and Degenerate Nucleic Acid
Ssequences
[0208] Modification and changes may be made in the sequence of the
proteins of the present invention and the nucleic acid segments
which encode them and still obtain a functional molecule that
encodes a protein with desirable properties. The following is a
discussion based upon changing the amino acid sequence of a protein
to create an equivalent, or possibly an improved, second-generation
molecule. The amino acid changes may be achieved by changing the
codons of the nucleic acid sequence, according to the codons given
in Table 9.
9TABLE 9 Codon degeneracies of amino acids Three Amino acid One
letter letter Codons Alanine A Ala GCA GCC GCG GCT Cysteine C Cys
TGC TGT Aspartic acid D Asp GAC GAT Glutamic acid E Glu GAA GAG
Phenylalanine F Phe TTC TTT Glycine G Gly GGA GGC GGG GGT Histidine
H His CAC CAT Isoleucine I Ile ATA ATC ATT Lysine K Lys AAA AAG
Leucine L Leu TTA TTG CTA CTC CTG CTT Methionine M Met ATG
Asparagine N Asn AAC AAT Proline P Pro CCA CCC CCG CCT Glutamine Q
Gln CAA CAG Arginine R Arg AGA AGG CGA CGC CGG CGT Serine S Ser AGC
AGT TCA TCC TCG TCT Threonine T Thr ACA ACC ACG ACT Valine V Val
GTA GTC GTG GTT Tryptophan W Trp TGG Tyrosine Y Tyr TAC TAT
[0209] Certain amino acids may be substituted for other amino acids
in a protein sequence without appreciable loss of enzymatic
activity. It is thus contemplated that various changes may be made
in the peptide sequences of the disclosed protein sequences, or
their corresponding nucleic acid sequences without appreciable loss
of the biological activity.
[0210] In making such changes, the hydropathic index of amino acids
may be considered. The importance of the hydropathic amino acid
index in conferring interactive biological function on a protein is
generally understood in the art (Kyte and Doolittle, J. Mol. Biol.,
157: 105-132, 1982). It is accepted that the relative hydropathic
character of the amino acid contributes to the secondary structure
of the resultant protein, which in turn defines the interaction of
the protein with other molecules, for example, enzymes, substrates,
receptors, DNA, antibodies, antigens, and the like.
[0211] Each amino acid has been assigned a hydropathic index on the
basis of its hydrophobicity and charge characteristics. These are:
isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine
(+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8);
glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9);
tyrosine (-1.3); proline (-1.6); histidine (-3.2);
glutamate/glutamine/aspartate/a- sparagine (-3.5); lysine (-3.9);
and arginine (-4.5).
[0212] It is known in the art that certain amino acids may be
substituted by other amino acids having a similar hydropathic index
or score and still result in a protein with similar biological
activity, i.e., still obtain a biologically functional protein. In
making such changes, the substitution of amino acids whose
hydropathic indices are within .+-.2 is preferred, those within
.+-.1 are more preferred, and those within .+-.0.5 are most
preferred.
[0213] It is also understood in the art that the substitution of
like amino acids may be made effectively on the basis of
hydrophilicity. U.S. Pat. No. 4,554,101 (Hopp, T. P., issued Nov.
19, 1985) states that the greatest local average hydrophilicity of
a protein, as governed by the hydrophilicity of its adjacent amino
acids, correlates with a biological property of the protein. The
following hydrophilicity values have been assigned to amino acids:
arginine/lysine (+3.0); aspartate/glutamate (+3.0.+-.1); serine
(+0.3); asparagine/glutamine (+0.2); glycine (0); threonine (-0.4);
proline (-0.5.+-.1); alanine/histidine (-0.5); cysteine (-1.0);
methionine (-1.3); valine (-1.5); leucine/isoleucine (-1.8);
tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4).
[0214] It is understood that an amino acid may be substituted by
another amino acid having a similar hydrophilicity score and still
result in a protein with similar biological activity, i.e., still
obtain a biologically functional protein. In making such changes,
the substitution of amino acids whose hydropathic indices are
within .+-.2 is preferred, those within .+-.1 are more preferred,
and those within .+-.0.5 are most preferred.
[0215] As outlined above, amino acid substitutions are therefore
based on the relative similarity of the amino acid side-chain
substituents, for example, their hydrophobicity, hydrophilicity,
charge, size, and the like. Exemplary substitutions which take
various of the foregoing characteristics into consideration are
well known to those of skill in the art and include: arginine and
lysine; glutamate and aspartate; serine and threonine; glutamine
and asparagine; and valine, leucine, and isoleucine. Changes which
are not expected to be advantageous may also be used if these
resulted in functional fusion proteins.
[0216] All of the compositions and/or methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the compositions and/or methods and in
the steps or in the sequence of steps of the methods described
herein without departing from the concept, spirit and scope of the
invention. More specifically, it will be apparent that certain
agents which are both chemically and physiologically related may be
substituted for the agents described herein while the same or
similar results would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the spirit, scope and concept of the invention.
10TABLE 10 RESTRICTION SITES FROM FIG. 3 ENZYME CUT SITE Notl 693
Xhol 702 BsaAl 1510 Rsrll 1722 Xhol 2170 Dral 2817 BsaAl 4975 Dral
5980 Dral 5999 BsaAl 7195 Dral 7677 Dral 7754 Bglll 8440 Rsrll 8998
Bglll 9296 Ascl 9851 SexAl 9917 BsaAl 9933 Sfil 10387 Sbfl 10535
EcoRl 10594
[0217]
11TABLE 11 RESTRICTION SITES FROM FIG. 4 ENZYME CUT SITE Notl 678
Xhol 687 BsaAl 1497 Rsrll 1709 Xhol 2157 Dral 2804 BsaAl 4924 Dral
5929 Dral 5948 BsaAl 7144 Dral 7626 Dral 7703 Bglll 8389 EcoRl
8413
[0218]
12TABLE 12 RESTRICTION SITES FROM FIG. 5 ENZYME CUT SITE BsaAl 411
Notl 878 Bglll 1541 EcoRl 1555 Smal 1573 Smal 2240 Srfl 2240 Notl
2244 Dral 3368 Dral 3387 Dral 4079
[0219]
13TABLE 13 RESTRICTION SITES FROM FIG. 6 ENZYME CUT SITE BsaAl 411
Notl 878 Bglll 1541 BsaAl 2185 EcoRl 3094 EcoRl 3126 Smal 3144 Smal
3811 Srfl 3811 Notl 3815 Dral 4939 Dral 4958 Dral 5650
[0220]
14TABLE 14 RESTRICTION SITES FROM FIG. 7 ENZYME CUT SITE BsaAl 411
Notl 878 Bglll 1541 BsaAl 2019 Sbfl 2150 BsaAl 2523 Sbfl 2789 EcoRl
3083 Smal 3101 Smal 3768 Srfl 3768 Notl 3772 Dral 4896 Dral 4915
Dral 5607
[0221]
15TABLE 15 RESTRICTION SITES FROM FIG. 8 ENZYME CUT SITE BsaAl 411
Notl 878 Bglll 1541 Sfil 2259 EcoRl 2603 EcoRl 2635 Smal 2653 Smal
3320 Srfl 3320 Notl 3324 Dral 4448 Dral 4467 Dral 5159
[0222]
16TABLE 16 RESTRICTION SITES FROM FIG. 9 ENZYME CUT SITE BsaAl 411
Notl 878 Bglll 1541 BsaAl 3070 EcoRl 3131 BsaAl 3183 Smal 4029 Srfl
4029 Notl 4033 Dral 5157 Dral 5176 Dral 5868
[0223]
17TABLE 17 RESTRICTION SITES FROM FIG. 10 ENZYME CUT SITE Notl 693
Hindlll 704 EcoRV 1241 Bglll 1356 Hindlll 1362 Hindlll 1374 Sphl
1678 Sfil 2118 Ncol 2166 EcoRl 2462 Smal 2480 BamHl 2486 Smal 3147
Notl 3151 Hindlll 3162 EcoRV 3699 Bglll 3814 Hindlll 3820 Hindlll
3832 Sphl 4136 BspHl 4138 Ncol 5005 EcoRl 5356 Smal 5374 BamHl 5380
Smal 6041 Notl 6045 Xhol 6054 Sphl 6963 Ncol 6990 Xhol 7522 BspHl
11293 BspHl 11793 Sphl 12986 Hindlll 13143 EcoRV 13677 Bglll 13792
Sphl 13971 Sphl 14061 Ncol 14066 EcoRV 14277 Ncol 14321 Bglll 14648
SexAl 15269 Sfil 15739 EcoRl 15946 BamHl 15964
[0224]
18TABLE 18 RESTRICTION SITES FROM FIG. 11 ENZYME CUT SITE Notl 693
Hindlll 704 EcoRV 1241 Bglll 1356 Sphl 1535 Sphl 1625 Ncol 1630
Apal 2508 EcoRl 2909 EcoRl 2941 Smal 2959 BamHl 2965 Smal 3626 Notl
3630 Hindlll 3641 EcoRV 4178 Bglll 4293 Sphl 4472 Sphl 4562 Ncol
4567 Sfil 5011 Ncol 5059 EcoRl 5355 EcoRl 5387 Smal 5405 BamHl 5411
Smal 6072 Notl 6076 Xhol 6085 Sphl 6994 Ncol 7021 Xhol 7553 BspHl
11324 BspHl 11824 Sphl 13017 Hindll 13174 EcoRV 13708 Bglll 13823
Sphl 14002 Sphl 14092 Ncol 14097 EcoRV 14308 Ncol 14352 Bglll 14679
SexAl 15300 Sfil 15770 EcoRl 15977 BamHl 15995
[0225]
19TABLE 19 RESTRICTION SITES FROM FIG. 12 ENZYME CUT SITE Notl 693
Bglll 1356 BsaAl 1834 Sbfl 1965 BsaAl 2338 Sbfl 2604 EcoRl 2898
Smal 2916 Smal 3583 Srfl 3583 Notl 3587 Bglll 4250 Sfil 4968 EcoRl
5312 EcoRl 5344 Smal 5362 Smal 6029 Srfl 6029 Notl 6033 Xhol 6042
BsaAl 6850 Rsrll 7062 Xhol 7510 Dral 8157 BsaAl 10315 Dral 11320
Dral 11339 BsaAl 12535 Dral 13017 Dral 13094 Bglll 13780 Rsrll
14338 Bglll 14636 Ascl 15191 SexAl 15257 BsaAl 15273 Sfil 15727
Sbfl 15875 EcoRl 15934
[0226]
20TABLE 20 RESTRICTION SITES FROM FIG. 13 ENZYME CUT SITE Notl 693
Hindlll 704 EcoRV 1241 Bglll 1356 Sphl 1535 Sphl 1625 Ncol 2497
Hindlll 2938 EcoRV 2946 EcoRl 2950 EcoRl 2982 Smal 3000 BamHl 3006
Smal 3667 Notl 3671 Hindlll 3682 EcoRV 4219 Bglll 4334 Sphl 4513
Sphl 4603 Ncol 4608 Sfil 5052 Ncol 5100 EcoRl 5396 EcoRl 5428 Smal
5446 BamHl 5452 Smal 6113 Notl 6117 Xhol 6126 Sphl 7035 Ncol 7062
Xhol 7594 BspHl 11365 BspHl 11865 Sphl 13058 Hindlll 13215 EcoRV
13749 Bglll 13864 Sphl 14043 Sphl 14133 Ncol 14138 EcoRV 14349 Ncol
14393 Bglll 14720 SexAl 15341 Sfil 15811 EcoRl 16018 BamHl
16036
[0227]
21TABLE 21 RESTRICTION SITES FROM FIG. 14 ENZYME CUT SITE Notl 693
Hindlll 704 EcoRV 1241 Bglll 1356 Sphl 1535 Sphl 1625 Ncol 1630
EcoRl 2946 SnaBl 2998 Ncol 3032 EcoRV 3179 BamHl 3183 Smal 3844
Notl 3848 Hindlll 3859 EcoRV 4396 Bglll 4511 Hindlll 4517 Hindlll
4529 Sphl 4833 BspHl 4835 Ncol 5702 EcoRl 6053 Smal 6071 BamHl 6077
Smal 6738 Notl 6742 Hindlll 6753 EcoRV 7290 Bgll 7405 Sphl 7584
Sphl 7674 Ncol 7679 Sfil 8123 Ncol 8171 EcoRl 8467 EcoRl 8499 Smal
8517 BamHl 8523 Smal 9184 Notl 9188 Xhol 9197 Sphl 10106 Ncol 10133
Xhol 10665 BspHl 14436 BspHl 14936 Sphl 16129 Hindll 16286 EcoRV
16820 Bglll 16935 Sphl 17114 Sphl 17204 Ncol 17209 EcoRV 17420 Ncol
17464 Bglll 17791 SexAl 18412 Sfil 18882 EcoRl 19089 BamHl
19107
[0228]
22TABLE 22 RESTRICTION SITES FROM FIG. 15 ENZYME CUT SITE Notl 693
Hindlll 704 EcoRV 1241 Bglll 1356 Sphl 1535 Sphl 1625 Ncol 1630
EcoRl 2946 SnaBl 2998 Ncol 3032 EcoRV 3179 BamHl 3183 Smal 3844
Notl 3848 Hindlll 3859 EcoRV 4396 Bglll 4511 Sphl 4690 Sphl 4780
Ncol 4785 EcoRl 6101 SnaBl 6153 Ncol 6187 EcoRV 6334 BamHl 6338
Smal 6999 Notl 7003 Hindlll 7014 EcoRV 7551 Bglll 7666 Hindlll 7672
Hindlll 7684 Sphl 7988 BspHl 7990 Ncol 8857 EcoRl 9208 Smal 9226
BamHl 9232 Smal 9893 Notl 9897 Hindlll 9908 EcoRV 10445 Bglll 10560
Sphl 10739 Sphl 10829 Ncol 10834 Sfil 11278 Ncol 11326 EcoRl 11622
EcoRl 11654 Smal 11672 BamHl 11678 Smal 12339 Notl 12343 Xhol 12352
Sphl 13261 Ncol 13288 Xhol 13820 BspHl 17591 BspHl 18091 Sphl 19284
Hindlll 19441 EcoRV 19975 Bglll 20090 Sphl 20269 Sphl 20359 Ncol
20364 EcoRV 20575 Ncol 20619 Bglll 20946 SexAl 21567 Sfil 22037
EcoRl 22244 BamHl 22262
[0229]
23TABLE 23 RESTRICTION SITES FROM FIG. 16 ENZYME CUT SITE Notl 678
Spel 685 BsaAl 693 SanDl 698 Rsrll 705 SexAl 711 Pacl 722 Sgfl 730
Sfil 741 Ascl 748 Sbfl 760 Smal 766 Srfl 766 Dral 774 Swal 774 Xhol
779 Dral 1426 BsaAl 3546 Dral 4551 Dral 4570 BsaAl 5766 Dral 6248
Dral 6325 Dral 6424 Pacl 7426 Dral 7887 BsaAl 8144 BsaAl 8164 Dral
8394 Bglll 8582 Rsrll 9140 Bglll 9438 Ascl 9993 SexAl 10059 BsaAl
10075 Sfil 10529 Sbfl 10677 EcoRl 10736
[0230]
24TABLE 24 RESTRICTION SITES FROM FIG. 17 ENZYME CUT SITE Notl 678
Spel 685 BsaAl 693 SanDl 698 Rsrll 705 SexAl 711 Pacl 722 Sgfl 730
Sfil 741 Ascl 748 Sbfl 760 Smal 766 Srfl 766 Dral 774 Swal 774 Xhol
779 Dral 1426 BsaAl 3546 Dral 4551 Dral 4570 BsaAl 5766 Dral 6248
Dral 6325 Dral 6424 Pacl 7426 Dral 7887 BsaAl 8144 BsaAl 8164 Dral
8394 Bglll 8582 EcoRl 8606
[0231]
25TABLE 25 RESTRICTION SITES FROM FIG. 18 ENZYME CUT SITE BsaAl 411
Notl 878 Dral 951 Pacl 1953 Dral 2414 BsaAl 2671 BsaAl 2691 Dral
2921 Bglll 3109 EcoRl 3123 Smal 3141 Smal 3808 Srfl 3808 Notl 3812
Dral 4936 Dral 4955 Dral 5647
[0232]
26TABLE 26 RESTRICTION SITES FROM FIG. 19 ENZYME CUT SITE BsaAl 411
Notl 878 Dral 951 Pacl 1953 Dral 2414 BsaAl 2671 BsaAl 2691 Dral
2921 Bglll 3109 BsaAl 3753 EcoRl 4662 Smal 4680 Smal 5347 Srfl 5347
Notl 5351 Dral 6475 Dral 6494 Dral 7186
[0233]
27TABLE 27 RESTRICTION SITES FROM FIG. 20 ENZYME CUT SITE Notl 878
Hindlll 889 Sphl 1041 Pacl 1953 BspHl 2613 BspHl 2736 Bglll 3109
Sphl 3288 Sphl 3378 Ncol 4250 Hindlll 4691 EcoRV 4699 EcoRl 4703
Smal 4721 BamHl 4727 Smal 5388 Notl 5392 BspHl 6477 BspHl 7485
BspHl 7590
[0234]
28TABLE 28 RESTRICTION SITES FROM FIG. 21 ENZYME CUT SITE Xhol 271
Dral 280 Swal 280 Smal 288 Srfl 288 Sbfl 298 Ascl 302 Sfil 316 Sgfl
326 Pacl 334 SexAl 338 Rsrll 346 SanDl 353 BsaAl 361 Spel 365 Notl
372 Dral 445 Pacl 1447 Dral 1908 BsaAl 2165 BsaAl 2185 Dral 2415
Spel 2609 Smal 2867 Sfil 3315 EcoRl 3608 Smal 3626 Smal 4293 Srfl
4293 Notl 4297 Spel 4304 BsaAl 4312 SanDl 4317 Rsrll 4324 SexAl
4330 Pacl 4341 Sgfl 4349 Sfil 4361 Ascl 4368 Sbfl 4380 Smal 4386
Srfl 4386 Dral 4394 Swal 4394 Dral 5388 Dral 5407 Dral 6099
[0235]
29TABLE 29 RESTRICTION SITES FROM FIG. 22 ENZYME CUT SITE BsaAl 411
Notl 878 Dral 951 Pacl 1953 Dral 2414 BsaAl 2671 BsaAl 2691 Dral
2921 Bglll 3109 BsaAl 4638 EcoRl 4699 BsaAl 4751 Smal 5597 Srfl
5597 Notl 5601 Dral 6725 Dral 6744 Dral 7436
[0236]
30TABLE 30 RESTRICTION SITES FROM FIG. 23 ENZYME CUT SITE ENZYME
CUT SITE ENZYME CUT SITE Notl 678 Spel 7388 Notl 11596 Hindlll 689
EcoRV 7413 BspHl 15339 Sphl 841 Sphl 7573 BspHl 15839 Pacl 1753
Smal 7646 Sphl 17032 BspHl 2413 Sfil 8094 Hindlll 17189 BspHl 2536
Ncol 8142 Sphl 17341 Bglll 2909 EcoRl 8387 Pacl 18253 Sphl 3088
Smal 8405 BspHl 18913 Sphl 3178 BamHl 8411 BspHl 19036 Ncol 3183
BamHl 9358 Bglll 19409 Apal 4061 EcoRl 9376 Sphl 19588 EcoRl 4462
BspHl 10162 Sphl 19678 Smal 4480 Ncol 10435 Ncol 19683 BamHl 4486
BamHl 10546 EcoRV 19894 Smal 5147 Ncol 10558 Ncol 19938 Notl 5151
Sfil 10569 Bglll 20265 Hindlll 5162 Sphl 10757 SexAl 20886 Sphl
5314 Bglll 10980 Sfil 21356 Pacl 6226 EcoRl 11052 EcoRl 21563 BspHl
6886 EcoRl 11455 BamHl 21581 BspHl 7009 Hindlll 11585
[0237]
31TABLE 31 RESTRICTION SITES FROM FIG. 24 ENZYME CUT SITE ENZYME
CUT SITE ENZYME CUT SITE Notl 678 BspHl 7050 Hindlll 11626 Hindlll
689 Spel 7429 Notl 11637 Sphl 841 EcoRV 7454 BspHl 15380 Pacl 1753
Sphl 7614 BspHl 15880 BspHl 2413 Smal 7687 Sphl 17073 BspHl 2536
Sfil 8135 Hindlll 17230 Bglll 2909 Ncol 8183 Sphl 17382 Sphl 3088
EcoRl 8428 Pacl 18294 Sphl 3178 Smal 8446 BspHl 18954 Ncol 4050
BamHl 8452 BspHl 19077 Hindlll 4491 BamHl 9399 Bglll 19450 EcoRV
4499 EcoRl 9417 Sphl 19629 EcoRI 4503 BspHl 10203 Sphl 19719 Smal
4521 Ncol 10476 Ncol 19724 BamHl 4527 BamHl 10587 EcoRV 19935 Smal
5188 Ncol 10599 Ncol 19979 Notl 5192 Sfil 10610 Bglll 20306 Hindlll
5203 Sphl 10798 SexAl 20927 Sphl 5355 Bglll 11021 Sfil 21397 Pacl
6267 EcoRl 11093 EcoRl 21604 BspHl 6927 EcoRl 11496 BamHl 21622
[0238]
32TABLE 32 RESTRICTION SITES FROM FIG. 25 ENZYME CUT SITE ENZYME
CUT SITE ENZYME CUT SITE Notl 678 EcoRl 9013 Sphl 15521 Hindlll 689
EcoRV 9246 Bglll 15744 Sphl 841 BamHl 9250 EcoRl 15816 Pacl 1753
Smal 9911 EcoRl 16219 BspHl 2413 Notl 9915 Hindlll 16349 BspHl 2536
Hindlll 9926 Notl 16360 Bglll 2909 Sphl 10078 BspHl 20103 Sphl 3088
Pacl 10990 BspHl 20603 Sphl 3178 BspHl 11650 Sphl 21796 Ncol 4050
BspHl 11773 Hindlll 21953 Hindlll 4491 Spel 12152 Sphl 22105 EcoRV
4499 EcoRV 12177 Pacl 23017 EcoRl 4503 Sphl 12337 BspHl 23677 Smal
4521 Smal 12410 BspHl 23800 BamHl 4527 Sfil 12858 Bglll 24173 Smal
5188 Ncol 12906 Sphl 24352 Notl 5192 EcoRl 13151 Sphl 24442 Hindlll
5203 Smal 13169 Ncol 24447 Sphl 5355 BamHl 13175 EcoRV 24658 Pacl
6267 BamHl 14122 Ncol 24702 BspHl 6927 EcoRl 14140 Bglll 25029
BspHl 7050 BspHl 14926 SexAl 25650 Bglll 7423 Ncol 15199 Sfil 26120
Sphl 7602 BamHl 15310 EcoRl 26327 Sphl 7692 Ncol 15322 BamHl 26345
Ncol 7697 Sfil 15333
[0239]
33TABLE 33 RESTRICTION SITES FROM FIG. 26 ENZYME CUT SITE ENZYME
CUT SITE ENZYME CUT SITE Bglll 649 EcoRl 6440 Dral 8098 Dral 1202
Smal 6712 BsaAl 8190 Dral 1278 Notl 6717 BsaAl 8731 BsaAl 1370 Spel
6724 Rsrll 8943 Sfil 2185 BsaAl 6732 EcoRl 9280 EcoRl 2529 SanDl
6737 Dral 10201 Smal 2801 Rsrll 6744 BsaAl 12321 Notl 2806 SexAl
6750 Dral 13326 Bglll 3468 Pacl 6761 Dral 13345 Dral 4021 Sgfl 6769
BsaAl 14541 Dral 4097 Sfil 6780 Dral 15023 BsaAl 4189 Ascl 6787
Dral 15100 Rsrll 4844 Sbfl 6799 Bglll 15786 Bglll 5142 Smal 6805
Dral 16339 Ascl 5697 Srfl 6805 Dral 16415 SexAl 5763 Dral 6813
BsaAl 16507 BsaAl 5779 Swal 6813 BsaAl 17248 Sfil 6233 Bglll 7469
EcoRl 18157 Sbfl 6381 Dral 8022
[0240]
34TABLE 34 RESTRICTION SITES FROM FIG. 27 ENZYME CUT SITE EcoRV 637
BglII 752 EcoRV 2829 HindIII 8420 BglII 9445 EcoRV 12082 HindIII
12086 BglIII 13111 EcoRV 15257 NotI 15268 BglII 16310 EcoRV 17613
BglII 17984 EcoRV 19548 NotI 19559
[0241]
35TABLE 35 RESTRICTION SITES FROM FIG. 28 ENZYME CUT SITE ENZYME
CUT SITE EcoRV 637 EcoRV 14937 EcoRV 2829 NotI 14948 HindIII 8420
EcoRV 17133 EcoRV 11922 EcoRV 19068 HindIII 11926 NotI 19079
[0242]
36TABLE 36 RESTRICTION SITES FROM FIG. 29 ENZYME CUT SITE ENZYME
CUT SITE ENZYME CUT SITE Notl 678 Ascl 748 BsaAl 3546 Spel 685 Sbfl
760 Dral 4551 BsaAl 693 Smal 766 Dral 4570 SanDl 698 Srfl 766 BsaAl
5766 Rsrll 705 Dral 774 Dral 6248 SexAl 711 Swal 774 Dral 6325 Pacl
722 Xhol 779 Bglll 7011 Sgfl 730 Dral 1426 EcoRl 7035 Sfil 741
[0243]
37TABLE 37 RESTRICTION SITES FROM FIG. 30 ENZYME CUT SITE ENZYME
CUT SITE ENZYME CUT SITE Xhol 271 BsaAl 361 SexAl 1771 Dral 280
Spel 365 Pacl 1782 Swal 280 Notl 372 Sgfl 1790 Smal 288 Smal 380
Sfil 1802 Srfl 288 Srfl 380 Ascl 1809 Sbfl 298 Smal 1047 Sbfl 1821
Ascl 302 EcoRl 1061 Smal 1827 Sfil 316 Bglll 1075 Srfl 1827 Sgfl
326 Notl 1738 Dral 1835 Pacl 334 Spel 1745 Swal 1835 SexAl 338
BsaAl 1753 Dral 2829 Rsrll 346 SanDl 1758 Dral 2848 SanDl 353 Rsrll
1765 Dral 3540
* * * * *