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

iGEM Nagahama

〜 Nagahama 〜

Specialty products of Nagahama

Food problems

Innovative food 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 ? ?

Experiment Experiment ２ Experiment １ Fragrance of garlic Fragrance of wasabi

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

Problem 香蔵庫“ KO-ZO-KO ” Muss cultivation To extract fragrance compounds To clear these subjects needs a huge cost and time

NEW KO-ZO-KO Flavorator 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 Medium does not touch source of fragrance ↓ this was closed and rapped ↓ left every medium for 21 hours

Experiment Effect of fragrance for growth of microbial A B Chassis: E. coli A,C: Control B: Farnesol D: Geraniol result C D

Experiment Effect of fragrance for growth of microbial Chassis: Bacillus subtilis ver. Natto A,C: Control B: Geraniol D: Farnesol result

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 Increasing STEP Ⅰ precursors STEP Ⅱ Production STEP Ⅲ 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

ispA λP L Increase the amount of Productio uction n for geraniol iol the Geraniol’s and F arnesol’s precursors idi ispDF dxs λP L marA λP L ObGES λP L Pr Production uction for Farneso esol m-ispA λP L dxs idi ispDF λP L marA λP L

Method How do we confirm the over producing of Geraniol and Farnesol ‘s precursors ?

Analysis of Ubiquinone-8 The difference intensity of light 90000 84718.4 80000 70000 Intensity of the band 60000 55343.56 50000 40000 30000 20000 10000 Left lane: IPTG (-) Right lane: IPTG (+) 0 IPTG minus IPTG plus

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

MEP Pathwa way glucose ispD dxs DXP MEP CDP-MEP CDP-ME Glyceraldehyde 3-phosphate ispF IPP idi HMBPP MEC DMAPP Pyruvic acid ispA m-is ispA ispA encodes farnesyl ispA ： FPP FPP GPP diphosphate synthase and geranyl diphosphate. It catalyzes IPP/DMAPP to GPP and FPP. Endogenous ObGES (Geraniol synthase) catalyzes IPP/DMAPP to m-is ispA ： phosphatase GPP It is mutant of ispA (S80F). Farnesol It lost function of farnesyl Geraniol iol diphosphate synthase(GPP to FPP) by mutation.

Farnesol production pathway Geraniol production pathway Precursor Precursor ispA m-ispA GPP GPP ispA ObGES Endogenous FPP phosphatase Geraniol Farnesol ispA catalyzes precursor to GPP and FPP. Farnesol is produced by Endogenous phosphatase.

MEP Pathwa way glucose ispD dxs DXP MEP CDP-MEP CDP-ME Glyceraldehyde 3-phosphate ispF IPP idi HMBPP MEC DMAPP Pyruvic acid ispA m-is ispA ispA encodes farnesyl ispA ： FPP FPP GPP diphosphate synthase and geranyl diphosphate. It catalyzes IPP/DMAPP to GPP and FPP. ObGES (Geraniol synthase) Endogenous catalyzes IPP/DMAPP to m-is ispA ： phosphatase GPP It is mutant of ispA (S80F). Geraniol iol Farnesol It lost function of farnesyl diphosphate synthase(GPP to FPP) by mutation.

Farnesol production pathway Geraniol production pathway Precursor Precursor ispA m-ispA GPP GPP ispA ObGES Endogenous FPP phosphatase Geraniol Farnesol m-ispA does not produce FPP. ispA catalyzes precursor to GPP and FPP. So, GPP is accumulated, and Farnesol is produced by Endogenous used to synthesize geraniol by phosphatase. ObGES .

ispA λP L dxs idi ispDF λP L λP L marA λP L ObGES λP L m-ispA dxs idi ispDF λP L λP L marA

Protocol of terpene production Decane phase Cultivate at 29 ℃ for 48h Use GC analyze ・ 2 × YT Medium Decane phase containing 1% absolve terpenes. glycerol. ・ Overlay decane on medium.

Result: 100 Farnesol production Ref Farnesol Peak intensity(%) 50 0 Retention time (min) Relative intensity (%) same reaction time Farnesol production device 100 Peak intensity (%) 50 0 4.0 8.0 Retention time (min) Relative intensity (%)

Result: Geraniol production “Which medium stronger than another one ?” But geraniol was not found in GC/MS 90% answered the medium B(WT) smelled analyzes… stronger than the medium A(recombinant).

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

Export 80 72.2 72.2 Protocol 70 Geraniol concentration (µg/ml) ↓ Culture (OD600=1.0) + 1.0% 60 geraniol solution 50 ↓ Cultivate at 30 ℃ overnight 42.9 42.9 ↓ Centrifugation 40 ↓ Wash the pellet with saline 30 solution ↓ Extract geraniol using chloroform 20 ↓ GC analysis 10 0 E. Coli JM109(WT) E. Coli JM109( marA ) E. coli JM109 (WT) E. coli JM109(marA) Overexpressing strain Fig : Intracellular geraniol concentration of E. coli decreased intracellular JM109 and its overexpressing of marA strain 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 ) Fig. :Comparison of colony numbers were spotted on LBGMg agar plates in addition of 0.5 %( v/v) geraniol solution serial ten- fold dilutions (10⁻¹～10⁻⁵), in the culture media overlaid with 1.0 % (v/v) geraniol hexane A: E. coli JM109 (WT) + hexane; B: E. solution (geraniol solution), and incubated coli JM109 ( marA ) + hexane; C: E. coli at 30 ° C for 24 h. JM109 (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 combination Plasmid’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

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

Innovative HP Questionnaire Education Cooperation News Letter Outreach & Dialogue

Outreach & Dialogue ! News ur project is… Advise ses Your Flaverator Gossips sips What at is this is? is cool!

But…. No money… Cro rowd wd Fu Funding ding

Crowd Funding ? USA .ver Japan .ver

Results & Discussion $24,000,000 About $ 37,000,000

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

Instructors & Advisers • Instructors Prof. Yoshisuke Nishi Prof. Shoji Usami • Advisers Koki Tsutsumi Shohei Takeshita Eishin Mitsui Ryuhei Minei Naoki Saito