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Result and Discussion

Confirm antibacterial activity of each volatile substances derived from plant

First, we examined our working hypothesis to “Flavorator” that the volatile gaseous substances from plants’ origin can show either the antibacterial or bacteriostatic activity in a box like “KOZOKO”. The results clearly showed that all the volatile substances of wasabi（Japanese horse radish）, rose, garlic and onion had antibacterial properties. In the literatures, wasabi, rose, garlic and onion have antibacterial volatiles, such as allyl isothiocyanete (wasabi), geraniol (rose), allicin (garlic), and lachrymaltory-factor (onion) These antibacterial volatiles are produced after complicated pathways, so for their syntheses, various enzymes are required Then, we searched metabolic pathways in E. coli, in which antibacterial volatiles can be either end products or intermediates. The search hit the geraniol. In E. coli, a precursor of geraniol, geranyl diphosphate (GPP), is synthesized. If we can successfully operate one enzyme to E. coli, it may synthesize geraniol using geranyl diphosphate as a precursor. In this context, we designed our system for establishing the concept of “Flavorator” to build up a brand-new biosynthetic pathways, in which geraniol is produced in the E. coli. In doing so, we transfer the three types of genes listed below to create the hyper-producer E. coli of geraniol.

We examined our working hypothesis to “Flavorator” that the volatile gaseous substances from plants’ origin can show either the antibacterial or bacteriostatic activity in a box like Kozoko.

Fragrance of Garlic

○Protocl here

Garlic grated produced its fragrances to suppress unwanted microbial growth. Left pork(A) didn't change the color. Right pork(B) changed Pink to Brown. We found from this result that fragrance of plants have antibacteril volatiles.

Left pork (A) was spread the garlic grated. Right pork (B) wasn’t spread the garlic grated. We left the two pork in the box at 18℃ for 2 month. Left pork (A) didn’t change the color. Right pork (B) changed Pink to Brown.

Fragrance of wasabi

Protocl here

Wasabi grated produced its fragrances to suppress unwanted microbial growth. We found from this result that fragrance of plants have antibacteril volatiles.

図の説明

Fragrance of Rose

Protocl here

Rose grated produced its fragrances to suppress unwanted microbial growth. We found from this result that fragrance of plants have antibacteril volatiles.

fig1：Storage of food by geraniol. A:ddH2O（300 μl）B:geraniol（stock solution, 300 μl） Incubation: 35 days Temperature: room temperature Chopsticks dipped in suspension of mold was put in the center of the bread. Mold were cultured among the left box(A), and mold weren’t cultured among the right box(B), suggesting that mold can’t be cultured in a state where geraniol is filled. This experiment indicate that geraniol might be the effect of suppressing the growth of bacteria.

fig2：Antibacterial confirmation of geraniol Comparison of the A and B（Chassis：Bacillus subtilis var. natto） A：ddH2O（300 μl）B：geraniol（stock solution, 300 μl） Incubation：21 hours Tempareture：37 ℃ Geraniol is released toward the center of the plate. Inhibition circle didn’t exist on the medium(A), and Inhibition circle existed on the medium(B), suggesting that Bacillus subtilis var. natto can't cultured at high concentration of geraniol. Comparison of the C and D（Chassis：E. coli） C：ddH2O（300 μl）D：geraniol（stock solution, 300 μl） Incubation：21 hours Tempareture：37 ℃ Geraniol is released toward the center of the plate. Inhibition circle didn’t exist on the medium(C), and Inhibition circle existed on the medium(D), suggesting that Bacillus subtilis var. natto can't cultured at high concentration of geraniol. Comparison of the B and D（Chassis：Bacillus subtilis var. natto and E. coli） Inhibition circle of Bacillus subtilis var. natto(B) is brighter than the inhibition circle of E. coli(D), suggesting that Bacillus subtilis var. natto is lower resistance to geraniol than E. coli. This experiment indicate that geraniol might be the effect of suppressing the growth of bacteria, and there might be a difference in the resistance of geraniol by bacteria.

fig3：Antibacterial confirmation of farnesol Comparison of the A and B（Chassis：E. coli） A：ddH2O（300 μl）B：farnesol（stock solution, 300 μl） Incubation：21 hours Tempareture：37 ℃ Farnesol is released toward the center of the plate. Inhibition circle didn’t exist on the medium(A), and Inhibition circle didn’t exist on the medium(B), suggesting that E. coli can cultured at high concentration of farnesol. Comparison of the C and D（Chassis：Bacillus subtilis var. natto） C：ddH2O（300 μl）D：farnesol（stock solution, 300 μl） Incubation：21 hours Tempareture：37 ℃ Farnesol is released toward the center of the plate. Inhibition circle didn’t exist on the medium(C), and Inhibition circle existed on the medium(D), suggesting that Bacillus subtilis var. natto can't cultured at high concentration of farnesol. Comparison of the B and D（Chassis：E. coli and Bacillus subtilis var. natto） Inhibition circle was only present on Medium of Bacillus subtilis var. natto(D), suggesting that Bacillus subtilis var. natto is lower resistance to farnesol than E. coli. This experiment indicate that farnesol might be the effect of suppressing the growth of bacteria, and there might be a difference in the resistance of farnesol by bacteria.

Increase in the amount of terpenoid's precursors

Analysis of ubiquinone-8 synthesized byE. coli JM109/BBa_K1653005
 by thin-layer chromatography (TLC) Right lane: IPTG Left lane: IPTG minus

Estimation of ubiquinone-8 content in spot Each intensity of spots indicating the content of ubiquinone-8

Analysis of ubiquinone-8 synthesized by E. coli JM109/BBa_K1653005  by thin-layer chromatography (TLC)
1. Put E. coli cells (JM109/MEP enzymes) in: methanol (7: 2).
2. Sonicate cells for 30 sec. and cool down 30 sec. and collect supernatants. ( 6 times).
3. Spin down the supernatants at 12,000xg, 10min at 4 ℃, and dry up acetone and methanol.
4. Add 400μl of chloroform : methanol ( 1 : 1 ) to the dried supernatants. Add equal volume 0.7% NaCl and spin down 12,000xg 5min at 4 ℃.
5. Extract the lower layer
6. Add 100μl of chloroform  : methanol (2: 1).
7. Spot the extracts on TLC plate and develop extracts using benzene : acetone (7 93).

Produce of Geraniol and Farnesol

Geraniol production

fig8:

fig9:

fig10:

Farnesol production

Farnesol production device [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1653025 (BBa_K1653025)]

λPL+r.b.s.+ispA＋MEP+×× (r.b.s.+dxs+r.b.s.+m-idi+r.b.s.+ispDF)

E. coli strain engineered with MEP pathway enzymes, ispDF, idi, and dxs , in combination with the enzyme gens, ispA, produced farnesol (Fig. 4B), which was detected by the Gas chromatography/Mas (Fig. 4A-G), having the same retention time as the farnesol chemica sample (Fig. 4A), while the counterpart control E. coli did not produce farnesol under the same conditions (Fig. 4C).　Neither E. coli engineered with MEP pathway enzymes only nor the one engineered ispA only showed any farnesol by the Gas chromatography/Mass (Figs. 4D and E). Farnesol is generated through hydrolysis of farnesyl diphosphate (FPP) by the endogenous phosphatases. Increase in farnesol should be associated with an increased intracellular FPP level. FPP is, in turn, converted from geranyl diphosphate (GPP), whose precursors are IPP and DMAPP. IPP and DMPP are end products of MEP pathway that exists in E. coli. Conversion to FPP from IPP or DMPP requires ispA (or m-ispA). Following this context, we speculate that E. coli could produce farnesol better than the counterpart control cells under the up-regulated cellular conditions of an increased intracellular MEP pathway enzymes by metabolic engineering in combination with the special enzyme that converts IPP or DMAPP into FPP.

Gas Chromatography/Mass(GC/MS)

Fig4:The FOH standard solution (Ref) was used as a control. The peak corresponding to the FOH standard at 8.5 min is indicated by an arrow. The peak at 8.5 min was applied to GC/MS. The FOH standard solution (Ref) was used as a control. E. coli JM109(Bba_K165025) were compared with respect to FOH formation using GC-MS. The fagment patarn is Similar with Ref.

ispA+MEP.dev[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1653025]

λPL+r.b.s.+ispA＋MEP+×× (r.b.s.+dxs+r.b.s.+m-idi+r.b.s.+ispDF)

FOH is probably generated through FPP hydrolysis by endogenous phosphatases, which are induced by an increased intracellular FPP level Analogously, we hypothesized that E. coli could produce FOH under cellular conditions of an increased intracellular FPP level through metabolic engineering. A MEP pathway has been shown to synthesize IPP and DMAPP efficiently in E. coli. Because of its high hydrophobicity and low volatility, decane was chosen to extract and solubilize FOH from culture broth. The decane overlay in the two-phase culture did not affect growth, and FOH could be solubilized in the decane phase with negligible volatile loss. We adopt 1 mL of decane overlaid to 5 mL of culture broth. Two-phase culture of E. coli JM109 (BBa_K165025) was carried out in 2YT medium containing 1% glycerol at 29°C for 48 h. The decane phase of the two-phase culture was collected to analyze the FOH content by GC-MS. In the GC-MS analysis (Fig. 4A-G), there was a main peak at 8.5 min in the collected decane phase sample, which corresponded to the reference solution of the standard FOH compound dissolved in decane. Mass spectrometry confirmed that the peak at 8.5 min was FOH (Fig. 4-A). However, the peak was not observed in two-phase culture without introducing BBa_K165025. The formation of FOH from FPP was further confirmed by blocking FPP synthesis. In the GC-MS, the FOH peak was observed in E. coli JM109 (BBa_K165025) culture, whereas no peak was observed with transformed E. coli JM109. It was found that FOH need not only ispA(BBa_K165018) but also MEP(BBa_K165024) in E. coli.We submit new part(BBa_K165025) as producing FOH.

Gas Chromatography/Mass(GC/MS)

Fig4:The FOH standard solution (Ref) was used as a control. The peak corresponding to the FOH standard at 8.5 min is indicated by an arrow. The peak at 8.5 min was applied to GC/MS. The FOH standard solution (Ref) was used as a control. E. coli JM109(Bba_K165025) were compared with respect to FOH formation using GC-MS.

Export of geraniol from E. coli

Export

[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1653020 BBa_K1653020]

Fig. XXX: Intracellular geraniol concentrations of E. coli JM109 (WT) and its overexpressing of marA strain, E. coli JM109 (marA).
In this figure, intracellular content of geraniol was less in the strain E. coli JM109 (marA) than the strain E. coli JM109 (WT). The concentrations of intracellular geraniol from E. coli JM109 (marA) was 42.9 μg/ml, which was 40% lower than that from of E. coli JM109 (WT), 72.2 μg/ml. This figure is suggesting that internalized geraniol could be more efficiently exported through AcrAB-TolC efflux pump following the presumed activation of this gene by introducing the activator marA gene.

Resistance

[http://parts.igem.org/wiki/index.php?title=Part:BBa_K1653020 BBa_K1653020]

Fig. X: 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 geraniol solutions, and incubated at 30°C for 24 h. This figure shows that E. coli JM109 (marA) cells that overexpress the marA product is more survived on geraniol overlay plates than the counterpart control E. coli JM109 wild type cells.

Fig. XX: Comparison of colony numbers after addition of geraniol solution. Time interval for treatment was set every 1 hour from 1 hour to 4 hours. A: E. coli JM109 (WT) + hexane; B: E. coli JM109 (marA) + hexane; C: E. coli JM109 (WT) + geraniol; D: E. coli JM109 (marA) + geraniol. As shown in Figs. XX A and B, treatment with hexane of E. coli JM109 (WT) and of E. coli JM109 (marA) showed similar colony numbers during these treatment intervals to those of time zero. This result suggests that hexane at this concentration and duration of time for 4hours did not affect both cell growth. In contrast, treatment with geraniol of E. coli JM109 (WT) and of E. coli JM109 (marA) showed toxicities to both strains (Figs. XX B, C and D). If we watch the colony numbers carefully, E. coli JM109 (marA) had more than E. coli JM109 (WT) during these treatment intervals ((Figs. XX C and D). These results demonstrate that toxicity of the geraniol was less to the strain E. coli JM109 (marA) than the strain E. coli JM109 (WT).