1. No category

How and why are saponins made in plants?
Departments of Plant- and Environmental Sciences
University of Copenhagen, Denmark
Soren Bak,
[email protected]
Department of Plant and Environmental Sciences
Natural products/Species specific metabolites
•Plants are the organic chemists per excellence in nature
•More than 200.000 different natural products known from
plants
•Enable plants to deter herbivores and pests, attract
pollinators, communicate with other plants and constantly
adapt to climatic changes
•A limited number of key gene families are constrantly
recruited for their biosynthesis.
Department of Plant and Environmental Sciences
Why are some types of Barbarea vulgaris
resistant to flea beetle herbivory and others not?
Barbarea vulgaris
(winter cress)
Pubescent
susceptible
Glabrous
resistant
Phyllotreta nemorum
(flea beetle)
Department of Plant and Environmental Sciences
Different geographical distributions of Barbarea
vulgaris - remnants of an ice age separation
G-type (Glabrous)
P-type (Pubescent)
Metabolite profiling: P versus G ecotype
- But which metabolites confer resistance???
Intens.
7
x10
Segregating
populations F1 & F2
4
P
x
3
F1
2
1
160
0
10
15
20
25
30
35
40
45
Time [min]
F2
G
Survival of flea beetle larvae on P, G, F1 & F2 plants
Segregating
populations F1 & F2
3
4
5
50
6
0
G1
G2
G3
G4
G5
G6
G7
G8
G9
G10
Bioassays with flea
beetle larvae
100
P1
P2
P3
P4
P5
P6
P7
P8
P9
P10
G
Larvae survived, %
P
x
Leaf
number
P and G
Plant number
15
160
F2
10
Number of plants
F1
F1
5
0
F2
40
20
0
≤ 10
11-30
31-50
51-70
71-90
Larvae survived, %
≥ 91
8
7
6
5
4
3
7
8
Correlation analysis and metabolite composition
identified saponins as the defensive compounds
Metabolite concentration ln(y+1)
4-Epihederagenin
cellobioside
Gypsogenin
cellobioside
Oleanolic acid
cellobioside
Hederagenin
cellobioside
1. m/z 819
2. m/z 817
3. m/z 803
4. m/z 819
Ret.time 42,5 min
Ret.time 42,0 min
Ret.time 46,1 min
Ret.time 39,6 min
Larvae survival ln(x+1)
oleanolic acid cellobioside
hederagenin cellobioside
gypsogenin cellobioside
4-epi-hederagenin cellobioside
LC-MS-NMR reveals a more complex saponin profile.
Intens.
x105
P type 277.cdf
5
4
6
7
4
19
9
2
3
10
8
18
11
14
3
12
17
15
16 20
21
2
1
13
1
40
45
50
55
60
65
70
Snap shot of time slice LC-MS-NMR experiment
75
Time [min]
Saponins are triterpenoid glycosides
•are prevalent in plants – but also occur in ancient animal
lineages.
Biological activities:
antifungal
antibacterial
molluscicidal
vermicidal
insecticidal
Pharmacologicaleffects:
anti-tumorigenic
anti-inflammatory
immunomodulatory
••••
••••
O
COOH
HO
O
diosgenin
oleanoli c acid
HO
Representatives of triterpene and steroidal sapogenins
Soap wort
(Saponaria officinalis)
DePartment of Plant and Environmental Sciences
Commercial use of saponins from camellia oleifera fruits.
Substitute antibiotics
Foaming agents
Camellia oleifera,–
tea seed oil plant
Earth worms
Slugs
Department of Plant and Environmental Sciences
Triterpene saponins of Quillaja
saponaria show strong aphicidal and
deterrent activity against the pea
aphid
soap bark tree
Quillaja saponaria
Induced mortality of aphids by Q.
saponaria after feeding on an
artificial diet
Department of Plant and Environmental Sciences
OSCs are the branch points in their biosynthesis
OSCs
OSCs have diverged to create new product profiles
from 2,3-oxidosqualene
100 BvLUP2-P
100
AtLUP2
95
[β-amyrin, taraxasterol + 4 minor]
AtLUP5
100
100
96
BvLUP2-G
[tirucalla-7,24-dien-3b-ol, isotirucallol + 4 minor]
BvLUP5-P
[α-amyrin, β-amyrin + 1 minor]
BvLUP5-G
[β-amyrin, α-amyrin]
AtLUP1
[lupeol, lupane-3b,20-diol + 4 minor]
AtLUP3 (CAMS1) [camelliol + 2 minor]
99
AtLUP4 (BAS)
[β-amyrin + 4 minor]
AtCAS1 [cycloartenol]
AtLAS1 [lanosterol]
100
0.1
cycloartenol
lupeol
Sterol
metabolism
AtPEN5 (MRN1) [marneral + 3 minor]
Specialized metabolism
93
[lupeol + 1 minor]
How are saponins made in plants?
1. cyclisation – oxidosqualene cyclases (OSCs)
2. oxidation - cytochrome P450 monooxygenases (P450s)
3. glycosylation - UDP-glycosyltransferases
(UGTs) acid
cycloartenol
oleanolic
OSC
gypsogenin
UGT #2
UGT #1
4-epi-hederagenin
lupeol
P450 #1
β-amyrin
3-O-β-D-glucopyranosyl-hederagenin
hederagenin
2,3-oxidosqualene
oleanolic
oleanolic
erythrodiol
hederagenin
aldehyde
cellobioside
acid
P450 #2
How saponins may interact with membranes
vesiculation
pore formation
membrane domain disruption
phospholipid
sterol
sphingolipid
saponin
saponin-sterol complex
Sapogenin 3-O-glucosylation by a neofunctionalized UGT73C
BvUGT73C9
99 BvUGT73C11*
100
BvUGT73C10*
B. vulgaris
BvUGT73C12
98
84 BvUGT73C13
AtUGT73C6
99
90
86
100
AtUGT73C5
AlUGT73C5
AtUGT73C2
BrUGT73C2
100
AlUGT73C3
66
60
AtUGT73C3
AtUGT73C1
98
AlUGT73C1
AtUGT73C7
100
BrUGT73C7
AtUGT73B5
0.1
models reveal positive
selection
AtUGT73C4
BrUGT73C1
100
*dN/dS site and branch
Bv: Barbarea vulgaris
At: Arabidopsis thaliana
Al: Arabidopsis lyrata
Br: Brassica rapa
Host range specificities for cabbage family insects
Potential for use of saponins as biopesticides?
Cabbage White
Green-veined White
Diamondback Moth
Flea Beetle
Pieris rapae
Pieris napi
Plutella xylostella
Phyllotreta nemorum
B. vulgaris arcuata G
+
-
-
-
B. vulgaris arcuata P
+
+
+
+
Structure Activity Relationships – what determines bioactivity?
hederagenin cellobioside
hederagenin
antifeedant effect
weak
antifeedant effect
oleanolic acid cellobioside
no
antifeedant
effect
Thure Pavlo Hauser
Phyllotreta nemorum
oleanolic acid
(Jens Kvist Nielsen)
Structure activity relationships:
3-O-β-D-glc hederagenin deter feeding
3-O-β-D-glc hederagenin
ST
60
50
40
30
20
10
0
0 nmol
3.75
nmol
15 nmol 60 nmol
70
consumption area (mm2)
consumption area (mm2)
70
3-O-β-D-glc oleanolic acid
ST
60
50
40
30
20
10
0
0 nmol
15 nmol 60 nmol
DePartment of Plant and Environmental Sciences
Antifungal activity towards Gaeumannomyces
graminis var. tritici (Take-all fungus)
Avenacin A1 (A1)
antifungal
Carbonyl-Avenacin A1 (CA)
inactive
(Geisler, … Bak, and Anne Osbourn, PNAS 2013)
Combinatorial biochemistry - Designing triterpenes
in Nicotiana using CPMV-HT
Agrobacterium
tumefaciens
buffer
GFP
Metabolite analysis using
TLC
GC-MS LC-MS
Department of Plant and Environmental Sciences
Identified OSCs show different product profiles
P-type
Susceptible
G-type
Resistant
E
Lupeol
LUP2
Prot. identity 98%
Prot. similarity 98%
Prot. identity 84%
Prot. similarity 92%
Prot. identity 83%
Prot. similarity 91%
LUP5
LUP2
Prot. identity 98%
Prot. similarity 98%
E
β-amyrin
E
LUP5
α-amyrin
DePartment of Plant and Environmental Sciences
- and oxidizing them with P450s
LUP2
G
P
β-am
6%
β-am
2%
TIC
COOH
Betulinic acid
+ CYP716A81
+ CYP716A80
lup
94%
Lup
98%
lup
3% ole.ac
7%
lup ole.ac
3% 7%
COOH
u5
23%
u5
26%
Ursolic acid
bet.ac
64%
β-am
2%
lup
7%
u4
40%
ole.ac
5%
β-am
1%
lup
6%
COOH
u5
26%
u5
24%
bet.ac
22%
bet.ac
67%
bet.ac
13%
ole.ac
3%
u4
51%
Oleanolic acid
DePartment of Plant and Environmental Sciences
- and oxidizing them with P450s
LUP5
G
α-am
12%
+ CYP716A81
TIC
lup
10%
β-am
5%
+ CYP716A80
urs.ac
11%
P
lup
5%
β-am
36%
β-am
78%
α-am
59%
α-am lup
1% 0%
u3
u5
2%
7%
β-am
3%
u3
14%
lup
1%
u2
20%
u4 u3
8% 9%
Ursolic acid
ole.ac
41%
α-am
3%
ole.ac
32%
COOH
u5
8%
ole.ac
74%
u5 β-am
11% 12%
Betulinic acid
α-am lup
7% 1%
urs.ac
26%
urs.ac
1%
COOH
u1
3%
urs.ac u5
3% 5%
ole.ac
10%
β-am
7%
α-am
12%
u4
5%
lup
1%
COOH
u1
11%
u3
36%
Oleanolic acid
u2
10%
Reconstruction of glycosylated triterpene
biosynthesis
LUP5-G
LUP5-G + CYP716A-G1
*
*
*
LUP5-G + CYP716A-G1
+ UGT73C11
14
18
22
*
*
Time [min]
3-O-β-glc oleanolic acid
26
30
Khakimov et al., 2015
DePartment of Plant and Environmental Sciences
Triterpenes For Commercialization
TriForC: A pipeline for the discovery, sustainable production and commercial
utilization of known and novel high-value triterpenes with new or superior
biological activities.
http://triforc.eu
DePartment of Plant and Environmental Sciences
TriForC is funded 6,8 mill Euro by European Community's Seventh Framework
Programme ([FP7/2007-2013] under grant agreement° 613692
TriForC can be divided into five scientific and technical
objectives:
• Identify new bioactive triterpenes for commercial
development as exemplified for pharmaceuticals and
agrochemicals.
• Elucidate Structure-Activity-Relationships (SAR) of
triterpenes through biological activity screenings.
• Constitute a genetic toolbox that will allow mimicking
‘rainforest’-like structural triterpene diversity in the
laboratory.
• Develop a metabolic engineering platform for rational
design of triterpenes for production in bioreactors.
• Develop and upscale plant-based bioreactors for
sustainable commercial production and biorefining of highvalue triterpenes
DePartment of Plant and Environmental Sciences
And how about Quiona and saponins?
From Orla Møller Petersen
From Sven-Erik
Prøve 1 and Prøve 2 Quinoa
Quinoa
Quinoa
Quinoa
Real (Bolivia) Titicaca (DK) Puno (DK) 124 (DK)
80% MeOH Extract
LC-MS/MS
80% MeOH Extract
80% MeOH Extract
Saponification pH<2
Saponification pH>10
GC-MS
LC-MS/MS
From waste to value
GC-MS
LC-MS/MS
Department of Plant and Environmental Sciences
Relative concentrations of 3 major sapogenins
Prøve 1
Unknow
n_1
26%
Oleanoli
c acid
18%
Prøve 2
Unknown_1
22%
Quin.Real (Bolivia)
Oleanolic
acid
20%
Unknown_1
22%
Oleanolic
acid
22%
Oleanolic acid
Hederag
enin
56%
Hederagenin
58%
Quin.Titicaca (DK)
Quin.PunoOleanolic
(DK)
acid
6%
Oleanolic
acid
17%
Unknown_1
34%
Hederagenin
56%
Quin.124 (DK)
Hederagenin
Unknown_1
33%
Oleanolic
acid
34%
Unknown_1
45%
Hederagenin
49%
Hederagenin
49%
(acidic hydrolysis)
Hederagenin
33%
Unknown 1
30
DePartment of Plant and Environmental Sciences
Relative concentrations of tentatively identified saponins
within quinoa samples from LC-MS/MS data
Prøve 1
P23
P22 6%
P21 0%
8%
P20
P19
0%
7%
P24 P1
6% 5%
P2
3%
P3
0%
P5
P6
4%
6%
P8
9%
P18
7%
P17
5% P16
6%
P1
4%
P4
0%
P7
0%
P9
9%
P15 P13
P10
7% 7%
P11 6%
P14 P12
0%
0% 0%
P23
P22 3%
0%
P20
0%
P21
10%
P19
7%
P24
6%
P3 P2
0% 0% P5
4%
P6
5%
P7
0%
P8
9%
P9
9%
P18
5%
P17
5%
P4
3%
Prøve 2
P16
6%
P15 P13
P10
8% 7%
P11 7%
P14 P12 0%
0%
0%
• A total of 24 saponins were detected.
• 2-4 sugar moieties
DePartment of Plant and Environmental Sciences
Conclusions and some perspectives
• Saponin composition is complex
• Saponins confer pest resistance – but is dependent on
structure
• Synthetic biology/metabolic engineering can be used to tailor
saponins for production in bioreactors
• Need to unravel Structure-Activity-Relationships for targeted
effect
Acknowledgements
Bekzod Khakimov
Pernille Østerbye Erthmann
Vera Kuzina
Jörg M. Augustin
Sylvia Drok
Carl Erik Olsen
Claus Ekstrøm
Jens Kvist Nielsen
Sven Bode Andersen
Thure Hauser
Søren Balling Engelsen
Tetsuro Shinoda
Ery Fukoshima
Toshiru Muranaka
Anne Osbourn JIC
Katrin Geisler JIC
Esben Halkjær Hansen - Evolva
Hanne Volpin – KeyGene
Torben Asp – University of Aarhus
Tim Newlin (cartoonist)
Members of TriForC
And many others
DePartment of Plant and Environmental Sciences
Screening extracts and compounds for bioactivities
Ido Korman, WP2
DePartment of Plant and Environmental Sciences
Using microalgae to produce triterpenes
Lupeol
Andrew Spicer, WP4