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Promoting Artemisinin Biosynthesis in Artemisia annua Plants by Substrate Channeling

2016; Elsevier BV; Volume: 9; Issue: 6 Linguagem: Inglês

10.1016/j.molp.2016.03.004

1674-2052

Junli Han, Zhen Wang, Selvaraju Kanagarajan, Mengshu Hao, Anneli Lundgren, Peter E. Brodelius,

Insect and Pesticide Research

Professor Youyou Tu, China Academy of Chinese Medical Sciences, Beijing, was awarded the 2015 Nobel Prize in Physiology or Medicine for "her discoveries concerning a novel therapy against malaria" (https://www.nobelprize.org/nobel_prizes/medicine/laureates/2015/press.html). This therapy is based on the sesquiterpenoid artemisinin, isolated from Artemisia annua. Artemisinin and its derivatives, administered in the form of artemisinin-based combination therapies (ACTs), are currently the best treatment for malaria. In the past decade, the study of artemisinin biosynthesis has advanced considerably and the biosynthetic pathway has been elucidated (Figure 1A ). A key enzyme of this pathway is amorpha-4,11-diene synthase (ADS), which is the first enzyme of the pathway converting farnesyldiphosphate (FDP) to the aliphatic sesquiterpene amorpha-4,11-diene. This intermediate has the right carbon skeleton for the biosynthesis of artemisinin. ADS is a rate-limiting enzyme with a very low turnover number, which is a general feature of terpene synthases. Numerous efforts have been made to enhance the production of artemisinin, including synthetic biology, plant genetic engineering, and breeding of plants that have a high yield of artemisinin. Some genes of artemisinin biosynthesis (Figure 1A) have been overexpressed in A. annua and increased levels of artemisinin have been reported. However, low artemisinin producers (LAPs) have been used in these studies as summarized in Supplemental Table 1. We have investigated a new strategy, i.e., substrate channeling, by overexpressing fused genes in two relatively high artemisinin-producing varieties (HAPs) of A. annua (var. Anamed and var. Chongqing) (cf. Supplemental Table 1). Substrate channeling is a process of directly transferring the product of one enzyme to the next enzyme of the pathway without equilibration with the bulk cytosol. Reaction rates can be significantly accelerated and therefore substrate channeling has great potential in metabolic engineering (Zhang, 2011Zhang Y.P. Substrate channeling and enzyme complexes for biotechnological applications.Biotechnol. Adv. 2011; 29: 715-725Crossref PubMed Scopus (218) Google Scholar). Enzyme complexes and multifunctional enzymes can be constructed through scaffolds or fusion of genes. Our group has previously reported on the fusion enzyme of farnesyldiphosphate synthase (FDS) and epi-aristolochene synthase (EAS), a sesquiterpene synthase involved in the biosynthesis of phytoalexins in tobacco (Brodelius et al., 2002Brodelius M. Lundgren A. Mercke P. Brodelius P.E. Fusion of farnesyldiphosphate synthase and epi-aristolochene synthase, a sesquiterpene cyclase involved in capsidiol biosynthesis in Nicotiana tabacum.Eur. J. Biochem. 2002; 269: 3570-3577Crossref PubMed Scopus (55) Google Scholar). The amount of epi-aristolochene produced in vitro by the fusion enzyme was considerably higher than when the two single enzymes were used. In this study, ADS, the first and rate-limiting enzyme of artemisinin biosynthesis, and FDS were fused. For constitutive or trichome-specific expression of the fusion gene, we used the CaMV35S or the recombinant promoter of amorphadiene 12-hydroxylase (CYP71AV1), respectively (Wang et al., 2013Wang H. Han J. Kanagarajan S. Lundgren A. Brodelius P.E. Trichome-specific expression of the amorpha-4,11-diene 12-hydroxylase (cyp71av1) gene, encoding a key enzyme of artemisinin biosynthesis in Artemisia annua, as reported by a promoter-GUS fusion.Plant Mol. Biol. 2013; 81: 119-138Crossref PubMed Scopus (54) Google Scholar). The substrate for ADS, i.e., FDP, is the precursor of many primary and secondary metabolites in the plant. The fused enzyme ADS-FDS will convert IDP + DMADP to amorpha-4,11-diene (shaded box in Figure 1A). The fused enzyme was constructed in the same way as reported for the EAS::FDS construct (Brodelius et al., 2002Brodelius M. Lundgren A. Mercke P. Brodelius P.E. Fusion of farnesyldiphosphate synthase and epi-aristolochene synthase, a sesquiterpene cyclase involved in capsidiol biosynthesis in Nicotiana tabacum.Eur. J. Biochem. 2002; 269: 3570-3577Crossref PubMed Scopus (55) Google Scholar). The ADS::FDS construct was introduced into a modified pCAMBIA 1381Z (Wang et al., 2011Wang H. Olofsson L. Lundgren A. Brodelius P.E. Trichome-specific expression of amorpha-4,11-diene synthase, a key enzyme of artemisinin biosynthesis in Artemisia annua L., as reported by a promoter-GUS fusion.Am. J. Plant Sci. 2011; 2: 619-628Crossref Google Scholar) in order to obtain two transformation vectors with different promoters, i.e., pCAMBIA-1381Z-M::pCYP71AV1::[ADS-FDS] (Figure 1B) and pCAMBIA-1381Z-M::pCaMV35S::[ADS-FDS] (Figure 1C). These vectors were used to transform two different varieties of A. annua (var. Chongqing and var. Anamed) using the procedure previously reported (Han et al., 2005Han J. Wang H. Ye H. Liu Y. Li Z. Zhang Y. Zhang Y. Yan F. Li G. High efficiency of genetic transformation and regeneration of Artemisia annua L. via Agrobacterium tumefaciens-mediated procedure.Plant Sci. 2005; 168: 73-80Crossref Scopus (63) Google Scholar). Twelve transgenic lines were analyzed by Southern blotting (Supplemental Figure 1), i.e., transgenic lines of A. annua var. Chongqing (CYP-701, CYP-702, CYP-703) and var. Anamed (CYP-301, CYP-302, CYP-303), transformed with the vector pCAMBIA-1381Z-M::pCYP71AV1::[ADS-FDS], and transgenic lines of var. Chongqing (35S-801, 35S-802, 35S-803) and var. Anamed (35S-401, 35S-402, 35S-403), transformed with the vector pCAMBIA-1381Z-M::pCaMV35S::[ADS-FDS]. A pair of primers giving a fragment (142 bp) spanning the fusion site of the fusion gene [ADS-FDS] was designed to analyze the transcript level of the fusion gene (Supplemental Table 2). The fusion gene, under the control of the CaMV35S promoter, was expressed at a much higher level than that controlled by the CYP71AV1 promoter (Figure 1D and 1E). Compared with the wild-type A. annua var. Chongqing, the transcript level of ADS was increased up to 24 and four times, respectively, when the CaMV 35S promoter and CYP71AV1 promoter were used, and in the Anamed variety the corresponding figures were 6.5 and 2 times, respectively. However, the transcript level of FDS was not increased to any great extent in any of the analyzed transgenic A. annua plants. The first enzyme of artemisinin biosynthesis, ADS, is specifically expressed in glandular secretory trichomes (GSTs) of A. annua (Wang et al., 2011Wang H. Olofsson L. Lundgren A. Brodelius P.E. Trichome-specific expression of amorpha-4,11-diene synthase, a key enzyme of artemisinin biosynthesis in Artemisia annua L., as reported by a promoter-GUS fusion.Am. J. Plant Sci. 2011; 2: 619-628Crossref Google Scholar), while FDS, whose product FDP is the substrate for the biosynthesis of various terpenoids such as sterols and sesquiterpenes, is constitutively expressed in plants. The transcript level of FDS is high in wild-type A. annua (Ct ≈ 20 for both varieties), and therefore there is only a subtle increase of the FDS transcript level since the fusion gene [ADS-FDS] was expressed at much lower levels. For both varieties, the recorded Ct values for the fusion gene were around 28 and 24 for the CYP71AV1 and CaMV35S promoter, respectively. The transcript level of ADS in wild-type A. annua var. Anamed (Ct ≈ 22) was higher than that in wild-type A. annua var. Chongqing (Ct ≈ 27), which may explain why the observed increase of the transcript level of ADS in transgenic var. Chongqing was significantly higher than in transgenic var. Anamed. The biosynthesis of artemisinin is located to GSTs of A. annua since enzymes of artemisinin biosynthesis, i.e., ADS, CYP71AV1, and DBR2, are specifically expressed in GSTs (Wang et al., 2011Wang H. Olofsson L. Lundgren A. Brodelius P.E. Trichome-specific expression of amorpha-4,11-diene synthase, a key enzyme of artemisinin biosynthesis in Artemisia annua L., as reported by a promoter-GUS fusion.Am. J. Plant Sci. 2011; 2: 619-628Crossref Google Scholar, Wang et al., 2013Wang H. Han J. Kanagarajan S. Lundgren A. Brodelius P.E. Trichome-specific expression of the amorpha-4,11-diene 12-hydroxylase (cyp71av1) gene, encoding a key enzyme of artemisinin biosynthesis in Artemisia annua, as reported by a promoter-GUS fusion.Plant Mol. Biol. 2013; 81: 119-138Crossref PubMed Scopus (54) Google Scholar, Jiang et al., 2014Jiang W. Lu X. Qiu B. Zhang F. Shen Q. Lv Z. Fu X. Yan T. Gao E. Zhu M. Chen L. Zhang L. Wang G. Sun X. Tang K. Molecular cloning and characterization of a trichome-specific promoter of artemisinic aldehyde Δ11(13) reductase (DBR2) in Artemisia annua.Plant Mol. Biol. Rep. 2014; 32: 82-91Crossref Scopus (27) Google Scholar). Our results showed that the dihydroartemisinic acid and artemisinin content of transgenic plants was increased significantly when the fusion gene [ADS-FPS] was specifically expressed in GSTs (Figure 1F and 1G). Artemisinin content was increased up to 2.5 and 2.0 times for var. Chongqing and var. Anamed, respectively, and the absolute content was around 2.5% dry weight in both varieties, which is a relatively high yield (cf. Supplemental Table 1). Compared with the CYP71AV1 promoter, the CaMV35S promoter was not so effective in improving the artemisinin content (Figure 1F and 1G) even though the expression levels of the fusion gene were much higher (Figure 1D and 1E). These results are explained by the fact that most of the recombinant fusion enzyme in this case is found in tissues where there is no artemisinin biosynthesis. In the case of the CYP71AV1 promoter, the recombinant fusion enzyme is located to trichomes where all the enzymes of artemisinin biosynthesis are expressed. The levels of artemisinic acid and arteannuin B (cf. Figure 1A) were only marginally increased in plants overexpressing the ADS-FDS gene (data not shown). Obviously the increased carbon flow in these transgenic high artemisinin-producing plants is directed toward dihydroartemisinic acid and artemisinin biosynthesis. DBR2, located at the branching point (cf. Figure 1A), is the key enzyme of this channeling, and it was recently shown that the promoters of the DBR2 gene of high artemisinin-producing varieties of A. annua including var. Chongqing and var. Anamed carry a unique nucleotide sequence believed to be involved in the upregulation of the DBR2 gene (Yang et al., 2015Yang K. Monafared S.R. Wang H. Lundgren A. Brodelius P.E. The activity of the artemisinic aldehyde Δ11(13) reductase promoter is important for artemisinin yield in different chemotypes of Artemisia annua L.Plant Mol. Biol. 2015; 88: 325-340Crossref PubMed Scopus (38) Google Scholar). It may be of interest to look at the kinetic properties of the two fused enzymes. In general, the kcat values for sesquiterpene synthases are very low in vitro ( 150 min−1) (Hemmerlin et al., 2003Hemmerlin A. Rivera S.B. Erickson H.K. Poulter C.D. Enzymes encoded by the farnesyl diphosphate synthase gene family in the Big Sagebrush Artemisia tridentata ssp. spiciformis.J. Biol. Chem. 2003; 278: 32132-32140Crossref PubMed Scopus (82) Google Scholar). FDS is a homodimer and we assume that a dimer of the fusion protein is formed in vivo. In the study on the fusion of FDS and EAS, it was reported that the Km value for FDP with the recombinant tobacco EAS and the two fusion enzymes EAS/FDS and FDS/EAS was 1.7, 1.6, and 2.6 μM, respectively, indicating that the dimerization does not affect the steady state kinetics of the fusion enzyme to any great extent (Brodelius et al., 2002Brodelius M. Lundgren A. Mercke P. Brodelius P.E. Fusion of farnesyldiphosphate synthase and epi-aristolochene synthase, a sesquiterpene cyclase involved in capsidiol biosynthesis in Nicotiana tabacum.Eur. J. Biochem. 2002; 269: 3570-3577Crossref PubMed Scopus (55) Google Scholar). The Km value for recombinant ADS has been reported to be 2.0 μM (Picaud et al., 2005Picaud S. Olofsson L. Brodelius M. Brodelius P.E. Expression, purification, and characterization of recombinant amorpha-4,11-diene synthase from Artemisia annua L.Arch. Biochem. Biophys. 2005; 436: 215-226Crossref PubMed Scopus (96) Google Scholar) and that of FDS 5.5, 7.1, and 1.6 μM for DMADP, IDP and GDP, respectively (Hemmerlin et al., 2003Hemmerlin A. Rivera S.B. Erickson H.K. Poulter C.D. Enzymes encoded by the farnesyl diphosphate synthase gene family in the Big Sagebrush Artemisia tridentata ssp. spiciformis.J. Biol. Chem. 2003; 278: 32132-32140Crossref PubMed Scopus (82) Google Scholar). We suggest that the fact that FDS exhibits a much higher turnover rate than ADS is beneficial for the overall conversion of IDP/DMADP to amorpha-4,11-diene. It is likely that due to the relatively high potential turnover capacity of the FDS and proximity of the two enzymes, the ADS of the fusion protein will experience a higher local FDP concentration than the native ADS present in the cytosol. It is likely that the ADS of the fusion enzyme operates at substrate saturation, which results in a maximum production of amorpha-4,11-diene by the fusion enzyme resulting in significantly increased artemisinin production. This work was supported by research grants from the Faculty of Health and Life Sciences, Linnaeus University and the Kronan Foundation to P.E.B.