2-2 Fragrance

Abstract

We designed apple fragrance expression device with MpAAT1^[1]. MpAAT1 is able to produce ester compounds with apple fragrance using some alcohols and Acetyl-CoA. Fig. 2-2-1 shows the outline of the designed device. We performed gas chromatography to confirm the production of the esters, and the results revealed that MpAAT1 successfully was able to produce 2-methylbutyl acetate and butyl acetate using 2-methyl butanol and butanol respectively (Fig. 2-2-2). 2-methylbutyl acetate and butyl acetate are known to smell Apple fragrance.

Fig. 2-2-1. Apple fragrance expression device.
Abbreviations : 2-methyl butanol (2-MeBuOH), butanol (BuOH), 2-methylbutyl acetate (2-MeBuOAc), butyl acetate (BuOAc).

Fig. 2-2-2.　Gas chromatography analysis of apple fragrance expression device.
(1) MpAAT1 + BuOH, (2) No MpAAT1 + BuOAc, (3) No MpAAT1 + BuOH, (4) MpAAT1 + 2-MeBuOH, (5) No MpAAT1 + 2-MeBuOAc, (6) No MpAAT1 + 2-MeBuOH, (7) MpAAT1 - 2-MeBuOH.
This work is done by Toshitaka Matsubara.

Introduction

MpAAT1 gene was isolated from Malus pumila (popular apple)^[2]. This gene is expressed in leaves, flowers and fruit of apple. The recombinant enzyme (MpAAT1) to produce esters involved in apple fragrance, utilize various alcohol as substrates such as straight chain (C3–C10), branched chain, aromatic and terpene alcohols. In addition, various kinds of CoA derivatives, such as acetyl-CoA, are used for producing esters by MpAAT1. Both alcohol and CoA derivative are required for successful ester synthesis. The major components for producing apple fragrance are butyl acetate and 2-methylbutyl acetate^[3]. We engineered apple fragrance expression device to produce these molecules by MpAAT1. This device is used to Artificial Cooperation System in our projects, when dying cells are recued by its counterpart, it gives an apple fragrance for a token of their gratitude.

Result

We transformed MpAAT1([http://partsregistry.org/Part:BBa_K395602 BBa_K395602]) on pSB6A1 along with pTrx6 into E.coli BL21 DE3, and cultured after addition of alcohols(2-MeBuOH or BuOH) as substrates. After 12 hours of incubation, we extracted organic soution layer from the culture and analyzed by gas chromatography (Fig. 2-2-2). Peaks of the esters producing apple fragrance (2-MeBuOAc or BuOAc) were detected.

Fig. 2-2-2. Gas chromatography analysis of apple fragrance expression device.
(1) MpAAT1 + BuOH, (2) No MpAAT1 + BuOAc, (3) No MpAAT1 + BuOH, (4) MpAAT1 + 2-MeBuOH, (5) No MpAAT1 + 2-MeBuOAc, (6) No MpAAT1 + 2-MeBuOH, (7) MpAAT1 - 2-MeBuOH.
This work is done by Toshitaka Matsubara.

(A) In (2), the peak of BuOAc is shown. Comparing with (1), the position of the peak is in the same position. From this result, we can see MpAAT1 converted BuOH into BuOAc. (B) In (3), we observed the peak of BuOH, which we are going to use as substrate.This graph doesn’t show peak in the same place with that of BuOAc in (1).This means BuOH used as substrate doesn’t contain BuOAc. (C) In the same way as (A), from comparison of (4) and (5), we can see 2-MeBuOAC is synthesized from 2-MeBuOH. (D) (6) shows the peak of 2-MeBuOH, though no peak of 2-MeBuOAc. Thus, 2-MeBuOH doesn’t contain 2- MeBuOAc. (E) In (7), no peak of ester is observed. This sample wasn’t added with substrates, so MpAAT1 without substrates didn’t produce esters. From these results, we can conclude that E.Coli with MpAAT1 is able to convert substrate alcohols into esters. ↑ どっちがいいですか ↓ (A)In (2), the peak of BuOAc is shown. Comparing with (1), the position of the peak is in the same position. From this result, we can see MpAAT1 converted BuOH into BuOAc. (B)In (3), we observed the peak of BuOH, which we are going to use as substrate. This graph doesn’t show peak in the same place with that of BuOAc in (1). This means BuOH used as substrate doesn’t contain BuOAc . (C)In the same way as 1., from comparison of (4)and (5), we can see 2-MeBuOAC is synthesized from 2-MeBuOH. (D)(6) shows the peak of 2-MeBuOH, though no peak of 2-MeBuOAc.Thus, 2-MeBuOH doesn’t contain2- MeBuOAc. (E)(7), no peak of ester is observed. This sample wasn’t added with substrate, so MpAAT1 without substrate didn’t produce esters. From these results, we can conclude that E.Coli with MpAAT1 is able to convert substrate alcohol into esters.

Discussion

From the result of the experiment above, we can conclude that engineered E.coli successfully converted alcohols added as substrates into esters. Moreover, we synthesized MpAAT1 and esters using E.coli BL21 DE3 as a chassis, which was reported to be implausible in former report(s)^[1]. In 2008, C.R. Shen and J.C. Liao^[4] succeeded in synthesizing butanol from E.coli. If we take advantage of this engineered E.coli, we could produce apple fragrance ester without the addition of substrate.

From the result of the experiment above, we can conclude that apple fragrance expression device was able to produce esters using alcohols added as substrates. Moreover, we synthesized MpAAT1 and esters using E.coli BL21 DE3 as a chassis, which was reported to be implausible in former report(s) ^[1]. In 2008, C.R. Shen and J.C. Liao^[4] succeeded in synthesizing butanol from E.coli. If we take advantage of this engineered E.coli, we could produce apple fragrance ester without the addition of substrate.

Materials and Methods

Strains of E. coli
E. coli BL21 DE3

Varieties of plasmid
MpAAT1 on pSB6A1
Trx on pACYC184

Substrate
Butanol (final 0.4%)
2-methyl butanol (final 0.2%)

Inducer
100 mM IPTG
20% arabinose

Solution
Undecane solution: undecane 10 μL + ether 990 μL

MpAAT1 expression construct.
We ordered the synthesis of MpAAT1([http://partsregistry.org/Part:BBa_K395602 BBa_K395602]) from Mr. GENE.
This artificial gene was annealed with vector pSB6A1 as MpAAT1 expression plasmid.
Moreover, we introduced pTrx6 into this expression plasmid to stabilize the MpAAT1 gene product.

MpAAT1 over expression conditions
Artificial gene has T7 promoter on the upstream of MpAAT1.
This promoter works by taking over T7 RNA polymerase from E. coli.
Therefore we utilized E. coli BL21 DE3 which has T7 RNA polymerase.
Furthermore, arabinose was added in culture to induce Trx which has arabinose-induced promoter.

E. coli BL21 DE3
E. coli BL21 DE3 express T7 RNA polymerase by IPTG induction.
Therefore we added 3 μL of 100 mM IPTG and 15 μL of 20% arabinose in LB culture in order to express MpAAT1 and Trx in E. coli BL21 DE3.

Expression of MpAAT1 recombinant protein in E. coli
O/N E. coli
→100-fold dilution
→2 hour fresh culture
→make up the OD₅₉₀ = 0.1 with LB culture
→add substrate，antibiotics and inducer
→O/N
→harvest (7,000×g, 3 min)
→collect supernatant solution
→separating liquid layer and oil layer with ether (shake supernatant solution 0.5 mL, ether 0.5 mL and undecane soln. 2 μL)
→derive oil layer from supernatant solution with liquid-liquid extraction
→GC analysis.

GC analysis
GC: SHIMADZU GAS CHROMATOGRAPH GC-14B
Column: J&W SCIENTIFIC, DB-17, Film thickness 0.25 μm, Column Dimensions 15 m × 0.320 mm, Temperature Limits 40°C to 280°C (300°C Program)
Conditions: column temperature 35°C, injector temperature 180°C, detector temperature 180°C
Sample was injected 5 μL.

reference

[1] Edwige J. F. Souleyre, FEBS Journal, 272, 3132–3144 (2005)
[2] Young H, J Sci Food Agric, 71, 329-336 (1996)
[3] [http://www.ncbi.nlm.nih.gov/nucleotide/52139952 GenBank accession number AY707098]
[4] Shota Atsumi, Metabolic Engineering, 10, 305–311 (2008)