iGEM Nagahama Nagahama Specialty products of Nagahama Food - - PowerPoint PPT Presentation

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Jul 30, 2023 148 likes •824 views

iGEM Nagahama Nagahama Specialty products of Nagahama Food problems Innovative food preservation method Flavorator Contents Flavorator Step1: Overproducing of IPP and DMAPP Step2:Converting IPP and DMAPP into

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iGEM Nagahama

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〜Nagahama〜

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Specialty products of Nagahama

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Food problems

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Innovative

food preservation method

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Flavorator

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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
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30 years ago…

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香蔵庫“KO-ZO-KO”

It can store food with the fragrance of the plants

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REALLY

Let‘s make KO-ZO-KO !

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Experiment

? ?

Grated plant flagrance At 18 degree for 2 months

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Fragrance of garlic

Experiment １ Experiment ２

Fragrance of wasabi

Experiment

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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!

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But

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香蔵庫“KO-ZO-KO” Problem

Muss cultivation To extract fragrance compounds To clear these subjects needs a huge cost and time

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Flavorator

NEW KO-ZO-KO

  • E. coli
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Which flagrance did we choose ?

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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

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Effect of fragrance for growth of microbial

D: Geraniol A,C: Control B: Farnesol Chassis: E. coli

C D A B

result

Experiment

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Effect of fragrance for growth of microbial

B: Geraniol A,C: Control D: Farnesol Chassis: Bacillus subtilis ver. Natto result

Experiment

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Experiment ↓ put bread and cotton in the case

↓ dipped in suspension of Penicillium

  • n 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

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Experiment

Geraniol may preserve food

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How does E. coli make flagrance? It makes flagrance along 3 steps.

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How to make

STEPⅠ STEPⅢ STEPⅡ

Increasing precursors Production Export

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Recombinant E. coli

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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
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step 1

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

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Metabolic pathway diagram of E. coli

IPP: isopentenyl pyrophosphate DMAPP: dimethylallyl pyrophosphate

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Productio uction n for geraniol iol

ispA

λPL λPL

ispDF idi dxs

λPL

marA

λPL

ObGES m-ispA

λPL λPL

ispDF idi dxs

λPL

marA

Pr Production uction for Farneso esol

Increase the amount of the Geraniol’s and Farnesol’s precursors

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Method

How do we confirm the over producing

  • f Geraniol and Farnesol ‘s precursors ?
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Analysis of Ubiquinone-8

55343.56 84718.4 10000 20000 30000 40000 50000 60000 70000 80000 90000

Intensity of the band

IPTG minus IPTG plus

The difference intensity of light

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

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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
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Step2 : Product terpene

Terpene’s Precursor Terpene

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SLIDE 35 Geraniol iol ObGES (Geraniol synthase) ispA glucose Pyruvic acid DXP MEP CDP-ME CDP-MEP MEC HMBPP dxs ispD ispF MEP Pathwa way Glyceraldehyde 3-phosphate GPP m-is ispA IPP idi DMAPP FPP FPP ispA ispA： m-is 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.

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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

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SLIDE 37 Geraniol iol ObGES (Geraniol synthase) ispA glucose Pyruvic acid DXP MEP CDP-ME CDP-MEP MEC HMBPP dxs ispD ispF MEP Pathwa way Glyceraldehyde 3-phosphate GPP m-is ispA IPP idi DMAPP FPP FPP ispA ispA： m-is 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.

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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.

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ispA λPL λPL marA λPL ispDF idi dxs λPL ObGES m-ispA λPL λPL ispDF idi dxs λPL marA

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Protocol of terpene production

・2×YT Medium containing 1% glycerol. ・Overlay decane

  • n medium.

Decane phase

Cultivate at 29℃ for 48h

Decane phase absolve terpenes. Use GC analyze

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SLIDE 41 Relative intensity (%) Relative intensity (%) Ref Farnesol 100 Peak intensity(%) 50 4.0 8.0 Peak intensity (%) 50 100 Retention time (min) Retention time (min)

Farnesol production device

same reaction time

Result: Farnesol production

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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…

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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
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Step 3: Export & Resistance

marA It activates of AcrAB-TolC multidrug efflux pump. We used this gene to export geraniol to extracellular quickly BBa_K1653020

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Export

10 20 30 40 50 60 70 80

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

Geraniol concentration (µg/ml) 42.9 42.9 72.2 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.

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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),

  • verlaid 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. coli JM109 (WT) + 0.5 % geraniol solution; D: E. coli JM109 (marA) + 0.5 % geraniol solution.

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Conclusion

We achieved

  • Terpene precursor mass production(✔)
  • Farnesol production(✔)
  • Geraniol Production(▲)…Questionn
  • Export of geraniol to extracellular(✔)
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Future work

We could produce geraniol and farnesol little. ・Change combination

Plasmid’s copy number (high or low)

Promotor strength (strong or weak) Ribosome binding site (strong or weak) ・MVA pathway ・Changing chassis

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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
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INNO NNOVA VATIV TIVE HU HUMA MAN PR N PRAC ACTI TICE CE (HP HP)

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Innovative HP

Education Questionnaire News Letter Cooperation Outreach & Dialogue

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Your ur project is… Advise

ses

Outreach & Dialogue

Flaverator is cool!

What at is this is?

Gossips sips

!

News

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But….

No money…

Cro rowd wd Fu Funding ding

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Crowd Funding ?

USA .ver Japan .ver

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Results & Discussion

About $ 37,000,000 $24,000,000

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AT ATTRI TRIBU BUTI TIONS ONS ＆ AC ACKN KNOW OWLE LEDGE DGEME MENT NTS

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Instructors & Advisers

  • Instructors
  • Prof. Yoshisuke Nishi
  • Prof. Shoji Usami
  • Advisers

Koki Tsutsumi Shohei Takeshita Eishin Mitsui Ryuhei Minei Naoki Saito

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Supporters

  • Ex

Experime riment ntal l support

  • rt

GC C and GC-MC MC Prof.Yasushi Kawai Associate Prof. shinnichi Sasaki Stati atist stics cs

  • Prof. Makoto Hasegawa

Associate Prof. Masahumi Shionyu Lecturer: Hayato Saigo Assistant stant staff Takashi Hamada Associate Prof. Toru Komiya Di Discussion ssion or Ad Advice ice Aya Imamura Ryota Takai Taro Nakagawa Off ffer er of the reagent nt or labwa ware

  • Prof. Atsushi Oshima
  • Prof. Tamio Mizukami
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Supporters

  • Businessli

esslike ke support

  • rt

The clerical staff of Nagahama Institute of Bio- Science and Technology Security guards Staff of Learning support Center Library and Information Technology Center

  • Hu

Human practi ctice ce support rt Prof.Sanji Matsusima Prof.Masanao Miwa

  • Hu

Human practi ctice ce support rt 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

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Supporters

  • Pe

Persona sonal l contr tribution ibution 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

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Sponsors

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Thank you for listening of our presentation

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QU QUES ESTI TION ONS

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Original genome gene of ispD and ispF from E. coli JM109