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Elucidation of the Mogroside pathway in Siraitia grosvenorii

Siraitia Project

Ari Schaffer Efraim Lewinsohn Kobi Tadmor

Yosi BurgerNurit Katzir

Adi Faigenboim

Elad Oren

Shiri Freilich

Shmuel Shen Marina Petreikov Lena Eselson Galil TzuriAyala Meir Kobi Zimbler Leena Taha Lior Nir

Hugo Gottlieb, NMR, Bar IlanRotem Sertchook, protein modellingShifra Ben-Dor, WIS,Promoter analyses

Rachel Davidovitz-RikanatiShahar Cohen Vitaly Portnoy Nadine Baranes

SWEETNESS“A major hedonic pleasure”

➢The need for sugar consumption is one of our

basic pleasures, continuously growing in a

coarse of the modern history.

➢Sugar consumption leads to obesity and other

health-related problems.

➢Sweet is more reinforcing and attractive than

cocaine or heroin

Acesulfame-K Alitame Aspartame Cyclamate Saccharin Sucralose Neotame

Natural sweeteners:

➢Sugars

➢Proteins

➢Small Secondary Metabolites

• Flavonoids

• Terpenoids

➢Non-sugar sweeteners , especially natural ones, are of rising interest to

consumers and to the food and health industries.

Center for the Genetic Enhancement of Cucurbit Fruit Quality

The Volcani Center- ARO Israeli Ministry ofAgriculture

Cucurbitaceae family

(118 genera 825 species)

CucumisC.sativus C.melo

(Cucumber) (melon) CucurbitaC. Pepo & C. maxima(Pumpkin & squash)

CitrullusCitrullus lanatu(watermelon)

Cucurbit fruits

Mogroside V

➢Mogroside V (250-times sweeter then sucrose) is

derived from mature fruit of Monk fruit, known as

Luo-han-guo (Siraitia grosvenorii, Cucurbitaceae).

Triterpene glycosides

➢Anti cancer and diabetes treatments➢insect attractants for environmentally safe

insect control

OH

O

O

O

O

OH

O

OH

OHOH

HO

O

OHHO

HO

O HO

O

OH

OH

OHHOHO

O OOH

OHOH

HO

Mogroside V

H3C

CH3

CH3

CH3 CH3

HO

H3CHO

OH

3

11

24H3C

25 CH3

OH

23

2223

Numbering of squalene and mogrol

glucosylation

glucosylation

Mogrol

2,3,22,23-diepoxysqualene

Hemiterpenes Monoterpenes

Farnesyl proteins

Sesquiterpenes (e.g. patchoulol)

FPP

Geranylgeranyl proteins

Polyterpenes(e.g. rubber)

DMAPPIPP

GPP

GGPP

DMAPP IPP

Phytoene

Triterpenes(e.g. α-tomatine, Mogrol…)

Monoterpenes (e.g. linalool)

Chlorophylls Tocotrienols

Plastoquinones

GPP

GGPPPhytol

Irregular Terpenes:

Anistomene

Isoprene

Methylbutenol

Cytokinins

HMBPP

Giberellines

SPPChlorophylls Tocopherolls

Phylloquinones

CytosolPlastid

Mitochondria

IPP

Ubiquinone

MVApathway

MEPpathway

PrenilatedFlavonoidsIrregular

Terpenes: Chresantemyl-PP

Lavandulyl

Bioactive Phytofluene Diterpenes

Zeta-Carotene

Neurosporene

Pro-Lycopene Lycopene

Me-CPP

CDP-MEP

CDP-ME

MEP

DXP

Pyruvate G3P

Mevalonate diphosphate

Mevalonate

HMG-CoA

Aceto-acetyl-CoA

2xAcetyl-CoA

PSY

HDR

GGPPS

DXS

Squalene

The Catalytic Difference of Oxidosqualene Cyclases (OSC)

cucurbitaceae

6224 Accurate Mass Time of Flight (TOF)1200 HPLC system

Agilent

ACQUITY QDa Detector

Alliance HPLC system Waters

MS-spectrum of cucurbitadienol

C30H50O, m/z=426.3861

0

.5

.5

.5

.5

.5

.5

.5

.5

x1408

77

66

55

44

33

22

11

0

(M4 +0 H9 ). 3 +8 [-2 H4 29 O]

42 (M7. +3 H92 )

+32

(M4 +4 K7 ). +34 [-H56 26 O]

46 (M5+.3 K57 )

+68

Counts vs. Mass-to-Charge (m/z)390400410420430440450460470480490500

+ SgCDS

Control + squaleneepoxidase

0.4

0.8

0.6

1.4

1.2

1

1.6

0.2

0

0.6

0.4

0.8

1.224,25-monoepoxy cucurbitadienol

1

1.6

1.4

3 4 5 6 7 11 12 13 14 15 16

Ion

coun

tX10

7(4

07-4

62D

a)

8 9 10Retention time (min)

1* 2 3

4

5 6

7

65

4

2

0.2 10

Model of CDS with 24,25 epoxy cucurbitadienolItkin et al., PNAS, 2016

The Siraitia CDS gene makes BOTH cucurbitadienol and 24,25-monoepoxycucurbitadienol

The Siraitia epoxide hydrolase genes can transform the 24,25 epoxide to 24,25 dihydroxy form

Itkin et al., PNAS, 2016

Mogroside Pathway

➢Mogroside V is derived

from bitter triterpene

aglycone Mogrol

➢The biosynthetic pathway

for Mogrol production

remained vague till now.HO

OH

OH

HO

MogrolMogroside V

➢Fruit metabolome in a course o the fruit ripening➢A whole genome sequencing of Siraitia combined

with an extensive transcriptomic analysis of

developing fruit

➢Genes identification based on homology and

expression profiles

➢Functional expression of candidate genes in yeast and

in planta

Research strategy to solve the biosyntheticpathway to sweet mogrosides

When looking at plant genomesCYPs and GTs

constitute the largest families of enzymes in plant

metabolism.

Therefore - a challenge!

Hydroxylation and glycosylation enzymes

“Cucurbitome” – site containing transcriptome and genome data of the

cucurbits project

* Established with the efforts of the “bioinformatics team”

Shiri Freilich, Adi Faigenboim and Elad Oren

Cucurbitadienol synthase (CDS)-first committed step to cucurbitacins and

sweet mogrosides.➢Single copy in Siraitia genome. Functional

expression in yeast and tobacco and tomato.

triterpenoid synthase

2,3-oxidosqualene to 2,3;22,23-diepoxysqualene

➢The Siraitia genome harbors 5 genes encodingsqualene synthase. Of these, two showed highexpression in the 15d fruit. The one, involvedin mogroside pathway yet to be verified.

CYP9

7

CYP706

0.5

Mining for CYP450 capable ofhydroxylation at C11, C24&C25

From 191 members of the

family ,nearly 50 candidate

CYP450s from the Siraitia

genome + transcriptome were

functionally expressed in

yeast. A CYP450 capableof

hydroxylating at C11 was

identified (NO C24&C25)


➢Eight members of the epoxide hydrolase (EPH) family, present in Siraitia

genome, were examined as candidates for the synthesis of the trans-24,25-

dihydroxy cucurbitadienol from the 24,25-epoxy-cucurbitadienol.

➢Heterologous expression in yeast –> EPH 1,2,3

have similar activity, resulting in accumulation

of 24,25-dihydroxycucurbitadienol

Epoxide hydrolase - EPH


100

90

80

70

60

50

40

30

20

10

0

M5

IM5

Sia

M4A

M3x

M2E

Mogrosides development in Siraitia

15 34 50 77 90 103

Fruit development (days after pollination)

sweet mogrosides in ripe fruit(M4, M5)

bitter mogrosides in developing fruit

(M1- M3)

Rela

tive

peak

abun

danc

e

Mining for UGTs that glucosylate mogrol and mogrosides

UGT91

UGT92

UGT89

UGT73

UGT87

UGT84

UGT74

UGT75

UGT76

UGT85

UGT71

UGT79 UGT72

UGT93

UGT94

269_1

UGT90

0.5

UGT720

UGT88 UGT82 UGT83

UGT78


Mining for UGTs that glucosylate mogrol

and mogrosides100

90

80

70

60

50

40

30

20

10

015 34 50 77 90 103

M5

IM5

Sia

M4A

M3x

M2E


0.6

0.4

0.2

0

0.8

1

M1E1

0

0.2

0.4

0.6

0.8

1

2Re

lativ

epe

akAr

ea x

10

UGT720-269-1 + Mogrol

M2E M1A

UGT720-269-4 + Mogrol

UGT85-269-1 UGT85-269-4

➢UGTs and functional expression (E. coli) of ~100 candidates we

M identified those, capable of glucosylating at C3 and C24, both primary

and branching glucosylationsENZYMES expressed in E. coli

Substrate UGT74-345-2 UGT75-281-2 UGT720-269-1 UGT720-269-4

M M1-E1 M1-E1 M1A1 M1-E1

M1-A1 M2-E M2-E M2-E M2-E

M1-E1 M2-E

M2-A1 M3x M3x M3x M3x

M2-A M3 M3 M3 M3

M3-A1 Sia

Retention time (min)

UGT720-269-1

UGT720-269-1


UGT branching enzymes

UGT720-269-1

UGT720-269-1

UGT720-269-1


The proposedUGT720-269-1

UGT720-269-1

pathway for mogroside biosynthesis inS. grosvenoriifruit.➢Epoxidations➢Hydroxidation➢Monooxygenation➢Successive glucosylations


Gene Clustering ?

➢Highly syntenous genomic

organization of “mogroside-

encoding genes” among the

Cucurbitaceae indicates that

gene clustering alone cannot

account for the regulation of

this metabolic pathway.

Novel gene duplications andfunctionalization ?

4 4 4

UGT720


Hierarchical clustering of the expression patterns of the members of the five gene

families responsible for mogroside biosynthesis

*

**

*

*

*15

DAA

34DA

A

50DA

A

77DA

A10

3DAA

Leav

es

Root

s

Stem


➢Novel pathway elucidated for Mogroside V in Siraitia grosvenorii fruit

➢Surprisingly EPH participates in hydroxylation of C-24 and C-25 on Mogrol skeleton

➢Highly syntenous genomic organization of “mogroside-encoding genes” in Cucurbitaceae: gene clustering alonecannot account for the regulation of mogoroside pathway.

Summary

Summary

➢Combining Metabolomics with Genetic Engineering to study novel metabolic pathway

➢Known facts (e.g. metabolic pathways genes are mostly in clusters) not always true and we still can see surprises (e.g. mogroside pathway)

➢Sometimes educated guess will not help, and hard work must be done (e.g. checking many genes to elucidate one from the pathway).

Still have time ?

ThankYou

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