Production of guaianolide sesquiterpene lactones in engineered yeast
Hikaru SEKI
Graduate School of Engineering, Osaka University

Research aims
Sesquiterpenoids are a group of terpenoids consisting of three isoprene units. Plants
produce a vast array of useful sesquiterpenoids from a common precursor, farnesyl
diphosphate (FPP). Guaianolide sesquiterpene lactones (GSLs), which are mainly found inplants, are a group of sesquiterpenoids characterized by a 5,7,5-ring structure containing alactone ring. GSLs exhibit a wide range of pharmaceutical properties including selectiveinhibition of acute myelogenous leukemia stem cells1). However, production of thesecompounds depends on their extraction from plants and suffers from low yields.
Alternatively, GSLs can be produced in microbial hosts using biosynthetic genes. In thisstudy, we aimed to identify enzymatic genes that can be used to reconstruct the GSL
biosynthetic pathway to synthesize the simplest form of GSL in the yeast, Saccharomyces
cerevisiae.
Methods
Using a combinatorial biosynthesis approach, we successfully synthesized 13-δ-guaianoic
acid, a potential biosynthetic intermediate of the GSL pathway, by transforming S. cerevisiaewith genes encoding four enzymes from different plant sources: Aquilaria crassna δ-guaiene
synthase AcC2 (a sesquiterpene synthase catalyzing the cyclization of FPP into asesquiterpene scaffold, δ-guaiene), Lactuca sativa CYP71AV3 (a cytochrome P450
monooxygenasecatalyzing
thehydroxylationof
δ-guaiene
toproduce
13-hydroxy-δ-guaiene), and two dehydrogenases from Artemisia annua, ADH1 and ALDH1,
which convert 13-hydroxy-δ-guaiene into 13-δ-guaianoic acid (Fig. 1). Therefore, tosynthesize the simplest form of GSL from δ-guaianoic acid, we aimed to identify the P450(s)that catalyze the 6- or 8-hydroxylation of 13-δ-guaianoic acid, which may promote
subsequent spontaneous lactone ring formation (Fig. 1). As a resource for candidate genemining, we selected the GSL-producing species Aquilaria sinensis because of its available
transcriptome information. To test for potential δ-guaiene oxidizing activity of the obtained
candidate P450s, transgenic yeast co-expressing AcC2 and each of the five candidate P450s

were generated, and the yeast culture extracts were analyzed by gas chromatography-mass
spectrometry (GC-MS).

Fig. 1. Engineered pathway for biosynthesis of the simplest form of guaianolide
sesquiterpene lactone (GSL) in transgenic yeast

Results
We mined candidate P450s from A. sinensis due to the availability of its transcriptome
sequencing data. Raw reads were downloaded from the Sequence Read Archive of three data
sets containing transcriptome data of differentially expressed genes under stress conditions:
1) different parts of a 5-year-old A. sinensis treated with formic acid2), 2) a callus under saltstress3), and 3) healthy and wounded stems4). All raw reads were mixed and assembled denovo using Trinity software. Five candidate P450s having greater than 45% amino acidsequence identity with known sesquiterpene oxidases and showing extremely highexpression in the formic acid-induced agarwood part (with a 300- to 1,600-fold increase intranscript levels) were selected. To test their potential δ-guaiene oxidizing activities, each of
the five candidate P450s was co-expressed with the δ-guaiene synthase gene in an FPP
high-producing yeast strain (EPY300), and the ethyl acetate extracts were analyzed byGC-MS. Among the five candidate P450s, two exhibited activity against δ-guaiene:
AsCYP-1 produced 13-hydroxy-δ-guaiene (compound 1 in Fig. 2), and AsCYP-2 produced
an unknown δ-guaiene oxide (compound 2 in Fig. 2). Based on its molecular mass,compound 2 was speculated to be a derivative of δ-guaiene oxidized at two different
positions. We also tested the activity of AsCYP-2 against 13-δ-guaianoic acid but no activity
was detected (data not shown).

Fig. 2. Gas chromatography-mass spectrometry (GC-MS) analysis of the products of
AcC2/candidate P450-expressing yeast
Ethyl acetate extracts of
AcC2/AsCYP-1-expressing yeast

yeast culture were analyzed by GC-MS.
produced 13-hydroxy-δ-guaiene (compound 1).

AcC2/AsCYP-2-expressing yeast produced an unknown δ-guaiene oxide (compound 2).

Conclusion
In this study, we identified five candidate P450s from A. sinensis transcriptome data andanalyzed their potential δ-guaiene and 13-δ-guaianoic acid oxidizing activities. However,none of the candidate P450s showed desirable activities (i.e., the ability to catalyze the 6- or
8-hydroxylation of 13-δ-guaianoic acid that may promote subsequent spontaneous lactone
ring formation). Although reconstruction of GSL pathways in yeast was not successful in thisstudy, we identified two P450s active against δ-guaiene. Since both P450s (AsCYP-1 and -2)
belong to the CYP71 family and sharing 48% amino acid sequence identity with each other,mining of additional candidates highly homologous to these P450s from GSL-producing
plants would be a promising approach to identify desirable enzymes for reconstructing theGSL pathway in yeast.

References
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