iGEM Nagahama

〜Nagahama〜

Specialty products of Nagahama

Food problems

Innovativefood preservation method

Flavorator

Contents

• 香蔵庫

• Flavorator

• Step1: Overproducing of IPP and DMAPP

• Step2:Converting IPP and DMAPP into

farnesol or geraniol

• Step3:Releasing the farnesol and geraniol

from the cells

• Human practice

30 years ago…

香蔵庫“KO-ZO-KO”

It can store food with the fragrance of the plants

REALLY

Let‘s make KO-ZO-KO !

Experiment

? ?

Grated plant

flagrance

At 18 degree

for 2 months

Fragrance of garlic

Experiment １ Experiment ２

Fragrance of wasabi

Experiment

Experiment

The fragrance of the plants has antibacterial activity

It can work at room temperature

It doesn’t need electric power

KO-ZO-KO may be

a practical and innovative food

preservation method!

But

香蔵庫“KO-ZO-KO”

Problem

Muss cultivation

To extract fragrance compounds

To clear these subjects needs a huge cost and time

Flavorator

NEW KO-ZO-KO

E. coli

Which flagrance did we choose ?

Experiment

↓ put culture of E. coli or Bacillus subtilis ver.

Natto into LB medium

↓ left the lid open for an hour to fix in the

medium

↓ put filter paper in the center of the lid

↓ put water, Geraniol or Farnesol into filter

paper

↓ this was closed and rapped

↓ left every medium for 21 hours

Medium does not touch source of fragrance

Effect of fragrance for growth of microbial

D: Geraniol

A,C: ControlB: Farnesol

Chassis: E. coli

C D

A B

result

Experiment

Effect of fragrance for growth of microbial

B: Geraniol

A,C: Control

D: Farnesol

Chassis: Bacillus subtilis ver.

Natto

result

Experiment

Experiment ↓ put bread and cotton in the case

↓ dipped in suspension of Penicillium

on center of the bread

↓ sank into Geraniol to the cotton in the case A.

And do the water to the cotton in the case B.

↓ put the lid on both cases and sealed up

↓ left these cases at room temperature for 35 days

Result

Experiment

Geraniol may preserve food

How does E. coli make flagrance?

It makes flagrance along 3 steps.

How to make

STEPⅠ

STEPⅢ

STEPⅡ

Increasing precursors

Production

Export

Recombinant E. coli

Contents

• 香蔵庫

• Flavorator

• Step1:Overproducing of IPP and DMAPP

• Step2:Converting IPP and DMAPP into

farnesol or geraniol

• Step3:Releasing the farnesol and geraniol

from the cells

• Human practice

step 1

Increasing in the amount of Geraniol’s and Farnesol’s precursors

Metabolic pathway diagram of E. coli

IPP: isopentenyl

pyrophosphate

DMAPP:

dimethylallyl

pyrophosphate

Production for geraniolispA

λPL

λPL

ispDFididxs

λPL

marA

λPLObGES

m-ispAλPL

λPL

ispDFididxs

λPLmarA

Production for Farnesol

Increase the amount of

the Geraniol’s and

Farnesol’s precursors

MethodHow do we confirm the over producing

of Geraniol and Farnesol ‘s precursors ?

Analysis of Ubiquinone-8

55343.56

84718.4

0

10000

20000

30000

40000

50000

60000

70000

80000

90000

Inte

nsi

ty o

f th

e b

and

IPTG minus IPTG plus

The difference intensity of light

Left lane: IPTG (-) Right lane: IPTG (+)

Contents

• 香蔵庫

• Flavorator

• Step1:Overproducing of IPP and DMAPP

• Step2:Converting IPP and DMAPP into

farnesol or geraniol

• Step3:Releasing the farnesol and geraniol

from the cells

• Human practice

Step2 : Product terpene

Terpene’s Precursor

Terpene

Geraniol

ObGES (Geraniol synthase)

ispA

glucose

Pyruvic acid

DXP MEP CDP-ME CDP-MEP

MECHMBPP

dxs ispD

ispF

MEP Pathway

Glyceraldehyde 3-phosphate

GPP

m-ispA

IPP

idi

DMAPP

FPP

ispA

ispA：

m-ispA：

Farnesol

Endogenous

phosphatase

encodes farnesyl

diphosphate synthase and

geranyl diphosphate.

It catalyzes IPP/DMAPP to

GPP and FPP.

catalyzes IPP/DMAPP to

GPP

It is mutant of ispA (S80F).

It lost function of farnesyl

diphosphate synthase(GPP

to FPP) by mutation.

Geraniol production pathway

Precursor

GPP

Geraniol

m-ispA

ObGES

ispA catalyzes precursor to GPP and FPP.

Farnesol is produced by Endogenous

phosphatase.

Farnesol

Precursor

GPP

FPP

ispA

ispA

Endogenous

phosphatase

Farnesol production pathway

Geraniol

ObGES (Geraniol synthase)

ispA

glucose

Pyruvic acid

DXP MEP CDP-ME CDP-MEP

MECHMBPP

dxs ispD

ispF

MEP Pathway

Glyceraldehyde 3-phosphate

GPP

m-ispA

IPP

idi

DMAPP

FPP

ispA

ispA：

m-ispA：

Farnesol

Endogenous

phosphatase

encodes farnesyl

diphosphate synthase and

geranyl diphosphate.

It catalyzes IPP/DMAPP to

GPP and FPP.

catalyzes IPP/DMAPP to

GPP

It is mutant of ispA (S80F).

It lost function of farnesyl

diphosphate synthase(GPP

to FPP) by mutation.

Geraniol production pathway

Precursor

GPP

Geraniol

m-ispA

ObGES

ispA catalyzes precursor to GPP and FPP.

Farnesol is produced by Endogenous

phosphatase.

Farnesol

Precursor

GPP

FPP

ispA

ispA

Endogenous

phosphatase

Farnesol production pathway

m-ispA does not produce FPP.

So, GPP is accumulated, and

used to synthesize geraniol by

ObGES.

ispAλPL

λPLmarA

λPLispDFididxs

λPLObGES

m-ispAλPL

λPLispDFididxs

λPLmarA

Protocol of terpene production

・2×YT Medium containing 1% glycerol.

・Overlay decane on medium.

Decane phase

Cultivate at 29℃ for 48h

Decane phase absolve terpenes.

Use GC analyze

Rela

tive

in

ten

sity (

%)

Rela

tive

in

ten

sity (

%)

RefFarnesol

100

0

Pe

ak in

ten

sity(%

)

50

4.0 8.0

Pe

ak in

ten

sity (

%)

0

50

100

Retention time (min)

Retention time (min)

Farnesol production device

same reaction time

Result:

Farnesol production

Result: Geraniol production

“Which medium stronger than another one?”

90% answered the medium B(WT) smelled

stronger than the medium A(recombinant).

But geraniol was not found in GC/MS analyzes…

Contents

• 香蔵庫

• Flavorator

• Step1:Overproducing of IPP and DMAPP

• Step2:Converting IPP and DMAPP into

farnesol or geraniol

• Step3:Releasing the farnesol and geraniol

from the cells

• Human practice

Step 3: Export & Resistance

marAIt activates of AcrAB-TolC multidrug efflux pump.

We used this gene to export geraniol to extracellular quickly

BBa_K1653020

Export

0

10

20

30

40

50

60

70

80

E. coli JM109 (WT) E. coli JM109(marA)

Ger

anio

l co

nce

ntr

atio

n (

µg

/ml)

42.9

72.2

E. Coli JM109(WT) E. Coli JM109(marA)

Fig : Intracellular geraniol concentration of E. coli JM109 and its overexpressing of marA strain

Protocol↓Culture (OD600=1.0) + 1.0% geraniol solution↓ Cultivate at 30℃ overnight↓ Centrifugation ↓ Wash the pellet with saline solution ↓ Extract geraniol using chloroform↓ GC analysis

Overexpressing strain decreased intracellular geraniol concentration.

Enhancement of geraniol tolerance

Fig. : Colony formation efficiencies of E. coli JM109 engineered with marA on

geraniol overlaid plates.E. coli JM109 and E. coli JM109 (marA)

were spotted on LBGMg agar plates in

serial ten-fold dilutions (10⁻¹～10⁻⁵),

overlaid with 1.0 % (v/v) geraniol hexane

solution (geraniol solution), and incubated

at 30°C for 24 h.

Fig. :Comparison of colony numbers

addition of 0.5 %( v/v) geraniol solution

in the culture media

A: E. coli JM109 (WT) + hexane; B: E. coli JM109 (marA) + hexane; C: E. coliJM109 (WT) + 0.5 % geraniol solution;

D: E. coli JM109 (marA) + 0.5 %

geraniol solution.

Conclusion

We achieved

• Terpene precursor mass production(✔)

• Farnesol production(✔)

• Geraniol Production(▲)…Questionn

• Export of geraniol to extracellular(✔)

Future work

We could produce geraniol and farnesol little.

・Change combinationPlasmid’s copy number (high or low)

Promotor strength (strong or weak)

Ribosome binding site (strong or weak)

・MVA pathway

・Changing chassis

Contents

• 香蔵庫

• Flavorator

• Step1:Overproducing of IPP and DMAPP

• Step2:Converting IPP and DMAPP into

farnesol or geraniol

• Step3:Releasing the farnesol and geraniol

from the cells

• Human practice

INNOVATIVE

HUMAN PRACTICE (HP)

Innovative HPEducationQuestionnaire

News Letter

Cooperation

Outreach & Dialogue

Your project is…Advises

Outreach & Dialogue

Flaveratoris

cool!

What is this?Gossips

!News

But…. No money…

Crowd Funding

Crowd Funding ?

USA .ver

Japan .ver

Results & Discussion

About $ 37,000,000

$24,000,000

ATTRIBUTIONS ＆

ACKNOWLEDGEMENTS

Instructors & Advisers

• Instructors

Prof. Yoshisuke Nishi

Prof. Shoji Usami

• Advisers

Koki Tsutsumi Shohei Takeshita

Eishin Mitsui Ryuhei Minei

Naoki Saito

Supporters

• Experimental support

GC and GC-MC

Prof.Yasushi Kawai

Associate Prof. shinnichi Sasaki

Statistics

Prof. Makoto Hasegawa

Associate Prof. Masahumi Shionyu

Lecturer: Hayato Saigo

Assistant staff

Takashi Hamada

Associate Prof. Toru Komiya

Discussion or Advice

Aya Imamura

Ryota Takai

Taro Nakagawa

Offer of the reagent or labware

Prof. Atsushi Oshima

Prof. Tamio Mizukami

Supporters

• Businesslike support

The clerical staff of

Nagahama Institute of Bio-

Science and Technology

Security guards

Staff of Learning support

Center

Library and Information

Technology Center

• Human practice support

Prof.Sanji Matsusima

Prof.Masanao Miwa

• Human practice support

Ryosuke Sibato

The staff of Academist

Shuichiro Takahashi

The staff of Leave a Nest

The mayor of Nagahama

The municipal officer of

Nagahama

The staff of NHK

Kazuto Andou

Supporters

• Personal contribution

Yohei Taga

Takashi Hata

Atsuko Kihara

Takeshi Ibuki

Yukitaka Saito

Takatsuru Nishikawa

Prof. Sanji Matsushima

Prof. Hiroaki Yamamoto

Prof. Makoto Hasegawa

Associate Prof. Kazuo Kamemura

Associate Prof. Atsuko Iwamoto

Hirofumi Wakabayashi

Prof.Masanao Miwa

Sponsors

Thank you for listening of our presentation

QUESTIONS

Original genome

gene of ispD and ispFfrom E. coli JM109