igem nagahama2015.igem.org/files/presentation/nagahama.pdf · geraniol obges (geraniol synthase)...
TRANSCRIPT
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
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!
香蔵庫“KO-ZO-KO”
Problem
Muss cultivation
To extract fragrance compounds
To clear these subjects needs a huge cost and time
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
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
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
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
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.
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
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