Part:BBa_K1448001

Yeast ɤ-terpinene generator

This device contains the region of the TEF1 yeast constitutive promoter, GST tag, ɤ-terpinene synthase coding site(BBa_K1448000), and CYC1 terminator. This device can constitutively express ɤ-terpinene synthase in yeast (introducing this device makes yeast cells express ɤ-terpinene synthase constitutively).

Characterization

Western-bloting analysis

(Fig.2 Western-bloting analysis of GST and d-limonene synthase (Lane2), ɤ-terpinene synthase (Lane3) and β-pinene synthase(Lane4) fusion proteins expressed in yeast. (A) The gel picture of SDS-PAGE and western-blot; Lanes: 1, p427-TEF; 2, p427-TEF-LS; 3, p427-TEF-ɤTPNS; 4, p427-TEF-βPINS. (B) vector map of plasmids. (C) The names and molecular weights of each mono-terpene synthase.)

Yeast cells carrying plasmids of p427-TEF-GST-monoterpenoid synthase were cultivated overnight in SD medium (0.67% yeast nitrogen base and 2% glucose with G418) 50mL at 28°C. Cells were collected and kept at -30˚C. Western blot analysis was carried out using anti-GST antibody and 12.5% polyacrylamide gel with SDS.

<Results>　

Western blot data (Fig.2) showed clear bands around 90kDa in the lanes of p427-TEF- monoterpenoid synthase but not in the lane of empty vector.

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In vivo detection of ɤ-terpinene using GC

Introduction

Since ɤ-terpinene is a volatile material, we expected that an amount of ɤ-terpinene were detected at the headspace of the culture in which the transformant with ɤ-terpinene generator is cultivated and tried the detection by GC analysis.

Fig.4 GC-analysis data

GC analysis ofɤ-terpinene in the culture

Materials and Methods

We cultivated the transformants containing ɤ-terpinene generator in p427-TEF or only empty p427-TEF in 50 mL SD medium (0.67% yeast nitrogen base and 2% glucose with G418) at 29˚C, 120 rpm for 24 h.

Analytic instrument:　Shimadzu GC14A

Column: GLScience　InertCap1 (15m　Length,0.25 mm I.D., 0.25mm film thickness)

Injection port 200C, Detector port 210 C

Detector: Flame Ionization Detector

Column Oven Temperature 40C/5min- 10C/min-200C/5min

Carrier Gas: Helium

Result

we uesd SPME　（SOLID PHASE MICROEXTRACTION） to catch ɤ-terpinene at the headspace of the culture. However, any amounts of ɤ-terpinene were not detected at the headspace of the culture (the exposure time is 30min).

Next we tried to catch ɤ-terpinene in the liquid phase of the culture using monotrap (the exposure time is 1h) and an amount of ɤ-terpinene was detected in the liquid phase of the culture.

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Growth curve

Fig.4 transformant: BY4742 containing ɤ-terpinene generator inserted in p427-TEF, control: BY4742 containing empty p427-TEF

Introduction : Various mono-terpenoids are known to have an anti-microbial property. In order to evaluate the anti-microbial property of -terpinene, we made the growth curve of yeast cells containing -terpinene generator and compare to that of yeast cells without -terpinene generator.

Method : We cultivated the transformants containing -terpinene generator in p427-TEF or only empty p427-TEF in 50 mL SD medium (0.67% yeast nitrogen base and 2% glucose with G418) at 29˚C, 120 rpm for 24 h. OD600 was measured every 2 h.

Result : Yeast cells containing ɤ-terpinene generator showed slower growth than cells containing empty p427-TEF, indicating that γ-terpinene inhibits the growth of yeast cells.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 204
  Illegal XhoI site found at 715
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]