Functional Analysis of Amorpha-4,11-Diene Synthase (ADS) Homologs from Non-Artemisinin-ProducingArtemisiaSpecies: The Discovery of Novel Koidzumiol and (+)-α-Bisabolol Synthases

Abstract

The production of artemisinin, the most effective antimalarial compound, is limited to Artemisia annua. Enzymes involved in artemisinin biosynthesis include amorpha-4,11-diene synthase (ADS), amorpha-4,11-diene 12-monooxygenase (CYP71AV1) and artemisinic aldehyde Δ(11)13 reductase (DBR2). Although artemisinin and its specific intermediates are not detected in other Artemisia species, we reported previously that CYP71AV1 and DBR2 homologs were expressed in some non-artemisinin-producing Artemisia plants. These homologous enzymes showed similar functions to their counterparts in A. annua and can convert fed intermediates into the following products along the artemisinin biosynthesis in planta These findings suggested a partial artemisinin-producing ability in those species. In this study, we examined genes highly homologous to ADS, the first committed gene in the pathway, in 13 Artemisia species. We detected ADS homologs in A. absinthium, A. kurramensis and A. maritima. We analyzed the enzymatic functions of all of the ADS homologs after obtaining their cDNA. We found that the ADS homolog from A. absinthium exhibited novel activity in the cyclization of farnesyl pyrophosphate (FPP) to koidzumiol, a rare natural sesquiterpenoid. Those from A. kurramensis and A. maritima showed similar, but novel, activities in the cyclization of FPP to (+)-α-bisabolol. The unique functions of the novel sesquiterpene synthases highly homologous to ADS found in this study could provide insight into the molecular basis of the exceptional artemisinin-producing ability in A. annua.

Identification and characterization of a novel sesquiterpene synthase, 4-amorphen-11-ol synthase, from <i>Artemisia maritima</i>

“…Column chromatography was performed using silica gel 60N (spherical, neutral), with particle sizes 63-210 µm (Kanto Chemical, Tokyo, Japan) or 10% AgNO 3 impregnated silica gel (Wako, Osaka, Japan). The amorpha-4,11-diene and koidzumiol standards were prepared as described previously (Muangphrom et al 2014(Muangphrom et al , 2016. α-Bisabolol was purchased from Tokyo Chemical Industry (Tokyo, Japan).…”

“…First-strand cDNA was synthesized using the PrimeScript RT-PCR Kit (Takara Bio Inc.) with 1 µg total RNA as a template. To obtain the full-length open reading frame (ORF) encoding AmAOS, RT-PCR was performed using primers 1 and 2 as described previously (Muangphrom et al 2016). The amplified product was cloned into pENTR/D-TOPO and sequenced.…”

“…Yeast harboring the pYES2/ CT empty vector (Invitrogen) was used as a negative control. Transgenic yeast cells were cultured as described previously (Muangphrom et al 2016) with minor modifications. Yeast cells were cultured in synthetic complete medium (2 ml) containing 2% glucose in the absence of histidine, methionine, and uracil (SC-His-Met-Ura) at 30°C with shaking at 180 rpm overnight before scale-up to 50 ml in the same culture medium and further incubated under the same condition for 1 day.…”

“…Therefore, to determine the key factor controlling the artemisinin-producing ability in A. annua, we previously analyzed the expression and enzymatic function of putative sesquiterpene synthases highly homologous to AaADS, a sesquiterpene synthase involved in artemisinin biosynthesis, in 13 non-artemisinin-producing Artemisia plant species. Among these, A. maritima L., a perennial plant widely distributed in the northern hemisphere and mountainous regions that exhibits several pharmaceutical properties, including anthelmintic, antimicrobial, anti-inflammatory, bronchodilatory, and spasmolytic activities (Irum et al 2015;Møller et al 2013;Shah et al 2011), expresses putative sesquiterpene synthases highly homologous to AaADS in both its leaves and roots (Muangphrom et al 2016). We previously identified a homologous enzyme expressed in A. maritima leaves as (+)-α-bisabolol synthase (AmBOS) (Muangphrom et al 2016).…”

“…Among these, A. maritima L., a perennial plant widely distributed in the northern hemisphere and mountainous regions that exhibits several pharmaceutical properties, including anthelmintic, antimicrobial, anti-inflammatory, bronchodilatory, and spasmolytic activities (Irum et al 2015;Møller et al 2013;Shah et al 2011), expresses putative sesquiterpene synthases highly homologous to AaADS in both its leaves and roots (Muangphrom et al 2016). We previously identified a homologous enzyme expressed in A. maritima leaves as (+)-α-bisabolol synthase (AmBOS) (Muangphrom et al 2016). Herein we analyzed the function of the other putative sesquiterpene synthase isolated from the roots of A. maritima.…”