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がん幹細胞選択的な細胞毒性を示す植物成分の微生物生産に向けた研究關光 （大阪大学大学院工学研究科）

Guaianolide Sesquiterpene Lactones（GSLs）は、5員環-7員環-ラクトン環が縮合した基本構造をもつ植 物セスキテ ルペノイドの一 群である。幾つかのGSL は急性 骨髄性 白血病 幹細胞 に選 択的な細胞毒 性を有する ことが報 告され ている。本研 究では 、組換 え酵母 におけるGSLs合 成経路の再 構築をめ ざし た。シ ャム ジンコウ由来δ-guaiene合成酵 素、レタ ス由 来CYP71AV3、Artemisia annua由来ア ルコー ルデヒ ドロゲ ナーゼ（ADH1）およびアルデヒドデヒドロゲナーゼ（ALDH1）遺伝子を酵母 に導 入しGSLsの 前駆体と なりうる δ-guaianoic acidを de novo 合成した。次に、δ-guaianoic acidの6位あるいは8位を水酸化し 分子内脱水 縮合 によるラクトン環 形成を誘導し うる酵素の候 補として、シ ナジン コウから6種のCYP71ファミリーP450を選抜し酵素機能を解析し た。そのうち3種がδ-guaieneに対する酸化活 性を示したが 、目的とするラクトン環形成 誘導活性については検出されなかった。従って、さらなる候 補酵素遺伝 子の探索が 必要である。
MVA/MEP pathway

CYP71AV3/
AsCYP-2

AcC2

AaADH/ALDH？

Aa: Artemisia annua
Simplest forms of GSL

Arglabin (Artemisia glabella):

Micheliolide (Michelia compressa):

antitumor agentof leukemia cells and tumorigenesis

a clinically significant and noteworthy

δ-guaiene

As: Aquilaria sinensis

13-δ-guaianoic acidsignificantly suppresses the growth

AsCYP-1Fig.1 Guaianolide sesquiterpene lactones (GSLs)

AsCYP-1

GSLs have a wide range of pharmaceutical properties,
represented by the ability to selectively inhibit acute
myelogenous leukemia stem cells.

11,12-epoxy-7-hydroxy-δ-guaiene

Fig.2 Reconstruction of GSLs pathways in yeast

Candidate gene mining

Enzyme activity assays(a)• Aquilaria sinensis (Agarwood, 沈香)
produces GSLs

EIC (220.0)

The raw RNA-seq data were obtained from NCBI database withaccession number SRP068230 (Ye et al.,2016)
candidate P450s

gene ID

White wood

Agarwood

AsCYP-1AsCYP-2
AsCYP-3
AsCYP-4
AsCYP-5
AsCYP-6

TR334210.45

TR33523

2.7

TR21946

0.09

TR33319
TR2224

0.95

TR336083.86

311.32823.86145.521263.763595.51618.54

Guaiene sesquiterpene lactone
isolated from Aquilaria sinensis

5.06

AcC2
AcC2+
AsCYP-2AcC2+AsCYP-3

FPKM (fra g me n ts p e r k i l o b a s e o f e x on p e r mi l l i on re a d s ma p p e d )FPKM (fragments per kilobase of exonper million reads mapped)• A GSL, thapsigargin, accumulates
in the roots of T. garganica.
Andersen et al. Plant Physiology (2017)

Thapsia garganica

Ac: Aquilaria crassna？

Reaction products

AcC2
TgCYP-1

2AcC2+
TgCYP-24 5(mono-hydroxy-δ-guaiene)

3

Desired reaction products

AcC2+
TgCYP-3

Thapsigargin

13-hydroxy-δ-guaien

1or66-hydroxy-δ-guaiene 8-hydroxy-δ-guaiene(b)
R T 1 3 .0 4This work

δ-guaiene

This work

13-hydroxy-δ-guaiene

1

R T 1 3 .0 2

R T 1 1 .3 32

R T 1 1 .3 5

R T 1 1 .4 64

R T 1 3 .0 3R T 1 2 .0 9536Fig. 4 GC/MS analysis of the product of AcC2/P450s/CPR-expressingyeast(a) Extracted ion chromatograms (EICs) at m/z 220. (b) Mass spectra of peaks 1-6.

•Fig. 3 Phylogeny of candidate P450 enzymes

This work

Mass spectra and retention times of Peaks 1-6 were different from those of
13-hydroxy-δ-guaiene, suggesting that peaks 1-6 are mono-hydroxylated δguaiene other than 13-hydroxy-δ-guaiene.

Conclusion

Future plan• 4 candidate P450s from A. sinensis and T. garganica showed activities against δ-guaiene.

• Structural determination of the reaction products.
• Functional analysis of candidate P450s against δ-guaianoic acid.

• 6 unknown reaction products were presumed to be mono-hydroxylated δ-guaiene:
they are likely todiffer in the position of hydroxy group from C-13.

FPP

AcC2 CYP71AV3 ADH1 ALDH1P450

6Hor

8OHorHHO• Mining of additional candidate P450s highly homologous to these P450s (sharing 40%
amino acid sequence identity) from GSL-producing plants would be a promisingapproach to identify desirable enzyme for reconstruction of GSLpathway in yeast.

δ-guaianoic acidhydroxy δ-guaiene

Lactonization
reaction

GSL

Method for analysis of candidates P450s

Yutaro YAMADA, Mami OSAWA and Hiromi TOMITA (0saka Univ.) contributed to the work