_________________________________________________________________________
MPS 587
MBioS571
Advanced Plant Biochemistry
PLANT TERPENOIDS: METABOLISM AND FUNCTION
Mark Lange
Institute of Biological Chemistry
Washington State University
Lecture 3
Outline
1.
1.1
1.2
1.3
1.4
Enzymatic Mechanisms
HMG-CoA Reductase
IPP Isomerase
Prenyltransferases
Terpene Synthases
2.
Terpene Biotechnology
3.
Selected References
_________________________________________________________________________
1.
Enzymatic Mechanisms
1.1
HMG-CoA Reductase
CYTOPLASM
Sterols
MVA
HMG-CoA
HMG-CoA
MVA
Phytoalexins
ER
Catalytic
domain
N-terminal
membrane
anchor
LUMEN
Glycosylation
site
Linker
Transmembrane
domain
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1.2
IPP Isomerase
BHR+
B|
HR
P P OH2C
HS
Ha
HS
P P OH2C
Hb
Hc
Ha
Hb
Hc+
B
1.3
B|
Prenyltransferases
Example: GPP synthase
O P P
O P P
HR
1.4
- PPi
- HR+
O P P
HS
HS
Terpene Synthases
Gene structure:
RR
DD(XX)D
Legend:
Plastidial targeting
sequence
Glycosyl hydrolaselike domain
Conserved insert
sequence (diterpene
synthases)
Active site domain
Example: Limonene synthase
(Colby et al. (1993) J Biol Chem 268: 23016-23024)
OPP
OPP
H
OPP
OPP
M2+
(2)
OPP
(3)
M2+
(1)
(1)
(1)
H
(1)
(2)
(3)
H
(3)
Myrcene
(2 %)
BM Lange
alpha-Pinene
(2 %)
(-)-Limonene
(94 %)
H
(3)
(4)
(4)
beta-Pinene
(2 %)
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Example: epi-Aristolochene synthase
(Facchini and Chappell (1992) Proc Natl Acad Sci USA 89: 11088-11092)
H
H+
H
OPP
H
(Starks et al. (1997) Science 277: 1815-1820)
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2.
Terpene Biotechnology
A
Glc
G3P
DXS
PEP
DXP
DMAPP
IDI
HMBPP
+ PPS
+
GGPPS
GGPP
crtB
crtI
Lyc
IPP
Pyr
ACP
NRI
B
IPP
O P P
+
O
t-GPPS
t-LS
L6H
Red
IDH
O P P
DMAPP
(-)-Limonene
(-)-Carvone
Secretion
(-)-Limonene
C
MK
PMK
MPDC
IPPI
FPPS
Mevalonate
ADS
Amorphadiene
O O
O
AACT
AcetylCoA
HMGS
HMGR
MK
PMK
MPDC
IPPI
FPPS
ADS
O
O
Amorphadiene
Artemisinin
Metabolic engineering approaches designed to enhance the production of isoprenoids.
(A) Metabolic control circuit engineered to respond to flux dynamics for lycopene synthesis in E. coli (Farmer and Liao,
2000); engineered enzymes (DXS, 1-deoxy-D-xyxulose 5-phosphate synthase; IDI, isopentenyl diphosphate isomerase;
GGPPS, geranylgeranyl diphosphate synthase; crtB, phytoene synthase; crtI, carotenoid isomerase; PPS,
phosphoenolpyruvate synthase) are shown in blue; the NRI protein (bold font) acts both as a sensor of acetyl phosphate
and as a controller to activate the expression of the PPS and IDI genes.
(B) Heterologous expression of the carvone biosynthetic pathway in E. coli (Carter et al., 2003); the abbreviations for
genes encoding recombinantly expressed proteins are: t-GPPS, geranyl diphosphate synthase (truncated to exclude
plastidial targeting sequence) from Abies grandis; t-LS, (-)-limonene synthase (truncated to exclude plastidial targeting
sequence) from Mentha spicata; (-)-limonene 6-hydroxylase from Mentha spicata; cytochrome P450 reductase from
Mentha x piperita; (-)-isopiperitenol (carvone) dehydrogenase from Mentha x piperita.
(C) Introduction of the MVA pathway into E. coli to enhance sesquiterpene production (Martin et al, 2003); the
expression constructs contained the following genes: AACT, acetoacetyl-CoA thiolase; HMGS, 3-hydroxy-3methylglutaryl synthase; HMGR, 3-hydroxy-3-methylglutaryl reductase; MK, mevalonate kinase; PMK,
phosphomevalonate kinase; MPDC, mevalonate diphosphate decarboxylase; IDI, isopentenyl diphosphate isomerase;
FPPS, farnesyl diphosphate synthase; ADS, amorpha 4,11-diene synthase; mevalonate was fed to E. coli cells when the
construct of the upper panel was used, whereas no external feeding was required when the construct shown in the lower
panel was employed.
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D.
Golden rice
For more information visit http://en.wikipedia.org/wiki/Golden_rice
Golden rice was created by transforming rice with three beta-carotene biosynthesis genes:
1. psy (photoene synthase)
2. lyc (lycopene cyclase) both from daffodil (Narcissus
pseudonarcissus), and
3. crt1 from the soil bacterium Erwinia uredovora
geranylgeranyl
The psy, lyc and crt1 genes were transformed into the nuclear genome and placed under the control of an
endosperm specific promoter, so that they are only expressed in the endosperm. The lyc gene transfomed into
the rice has a transit peptide sequence attached so that it is targeted to the plastid where geranylgeranyldiphosphate formation occurs. It was important to use the bacterial gene crt1 since it can catalyze multiple
steps in the synthesis of carotenoids, these steps require more than one enzyme in plants. The end product of
the engineered pathway lycopene, if the plant accumulated lycopene the rice would be red. Recent analysis
has shown that the plant endogenous enzymes process the lycopene to beta-carotene in the endosperm,
giving the rice the distinctive yellow colour for which it is named. The original Golden rice was called SGR1,
under greenhouse conditions it produced 1.6µg/g of carotenoids.
In 2005 a team of Reseachers at biotechnology company Syngenta produced a variety of golden rice called
golden rice 2. They combined a photoene synthase gene from maize with the lyc and crt1 from the original
golden rice. Golden rice 2, produces 23 times more carotenoids than golden rice (up to 37µg/g), and
preferentially accumulates beta-carotene (up to 31µg/g of the 37µg/g of carotenoids). To receive half the
Recommended Dietary Allowance (RDA), it is estimated that 72 g of this rice would need to be eaten.
Golden rice has been bred with local rice cultivars in the Philippines, Taiwan and with the American rice variety
Cocodrie, the first field trials of these golden rice cultivars were conducted by Louisiana State University
AgCenter in 2004. Field testing will allow more accurate measurment of the nutritional value of golden rice and
will enable feeding tests to be performed. Preliminary results from the field tests shown that field grown Golden
rice produced 3 to 4 times more beta-carotene than the Golden rice grown under greenhouse conditions.
In June 2005, researcher Peter Beyer received funding from the Bill and Melinda Gates Foundation to further
improve Golden rice by increasing levels of or bioavailability of pro-vitamin A, vitamin E, iron, and zinc, and
improve protein quality though genetic modification.
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3.
Selected References
Bohlmann et al. (1998) Proc Natl Acad Sci USA 95: 4126–4133.
Croteau R (2000) In Biochemistry and Molecular Biology of Plants (Buchanan, Gruissem, Jones, Eds.) Am
Soc Plant Biol, Waldorf, MD, pp. 1250-1268.
Ye et al. (2000) Science 287: 303-305.
Farmer W.R. and Liao J.C. (2000) Nat Biotechnol 18: 533-537.
Liang et al. (2002) Eur J Biochem 269: 3339-3354.
Carter et al. (2003) Phytochemistry 64: 425-433.
Martin et al. (2003) Nat Biotechnol 21: 796-802.
Benveniste (2004) Annu Rev Plant Biol 55: 429-457.
Notes
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