Trichome-Specific Expression of Amorpha-4,11-Diene Synthase, a Key Enzyme of Artemisinin Biosynthesis in <i>Artemisia annua</i>L., as Reported by a Promoter-GUS Fusion

Artemisia annua L. produces small amounts of the sesquiterpenoid artemisinin, which is used for treatment of malaria. A worldwide shortage of the drug has led to intense research to increase the yield of artemisinin in the plant. In order to study the regulation of expression of a key enzyme of artemisinin biosynthesis, the promoter region of the key enzyme amorpha-4,11-diene synthase (ADS) was cloned and fused with the β-glucuronidase (GUS) reporter gene. Transgenic plants of A. annua expressing this fusion were generated and studied. Transgenic plants expressing the GUS gene were used to establish the activity of the cloned promoter by a GUS activity staining procedure. GUS under the control of the ADS promoter showed specific expression in glandular trichomes. The activity of the ADS promoter varies temporally and in old tissues essentially no GUS staining could be observed. The expression pattern of GUS and ADS in aerial parts of the transgenic plant was essentially the same indicating that the cis-elements controlling glandular trichome specific expression are included in the cloned promoter. However, some cis-element(s) that control expression in root and old leaf appears to be missing in the cloned promoter. Furthermore, qPCR was used to compare the activity of the wild-type ADS promoter with that of the cloned ADS promoter. The latter promoter showed a considerably lower activity than the wild-type promoter as judged from the levels of GUS and ADS transcripts, respectively, which may be due to the removal of an enhancing cis-element from the ADS promoter. The ADS gene is specifically expressed in stalk and secretory cells of glandular trichomes of A. annua.

Cloning, E. coil Expression and Molecular Analysis of Amorpha-4,11-Diene Synthase from a High-Yield Strain of Artemisia annua L.

increasing demand of artemisinin in the treatment of malaria has placed substantial stress on the total artemisinin supplies world-wide, so more attention has been paid to increasing the content of artemisinin in the Artemisia annua L. plant. In this study, amorpha-4, 11-diene synthase （ADS） cDNA （ads1） and genomics gene （gads1） were cloned from a high-yield A. annua strain 001. The activity of ADS1 was confirmed by heterogeneous overexpression of ads I and in vitro enzymatic incubation. Reverse transcript-polymerase chain reaction results demonstrated that ads1 expressed in leaves, flowers and young stems, but not in roots. This organ-specific expression pattern of ads1 is consistent with that of artemisinin accumulation in the plant. The gads1 has a complex organization including seven exons and six introns, and belongs to class III terpene synthase. DNA gel blotting revealed that the ADS gene has at least four copies in the genome of strain 001. The higher copy numbers might be one of the reasons for its high artemisinin content.

Amorpha-4,11-diene synthase: a key enzyme in artemisinin biosynthesis and engineering

Amorpha-4,11-diene synthase(ADS)catalyzes the first committed step in the artemisinin biosynthetic pathway,which is the first catalytic reaction enzymatically and genetically characterized in artemisinin biosynthesis.The advent of ADS in Artemisia annua is considered crucial for the emergence of the specialized artemisinin biosynthetic pathway in the species.Microbial production of amorpha-4,11-diene is a breakthrough in metabolic engineering and synthetic biology.Recently,numerous new techniques have been used in ADS engineering;for example,assessing the substrate promiscuity of ADS to chemoenzymatically produce artemisinin.In this review,we discuss the discovery and catalytic mechanism of ADS,its application in metabolic engineering and synthetic biology,as well as the role of sesquiterpene synthases in the evolutionary origin of artemisinin.

Allele-aware chromosome-level genome assembly of Artemisia annua reveals the correlation between ADS expansion and artemisinin yield

Artemisia annua is the major natural source of artemisinin,an anti-malarial medicine commonly used worldwide.Here,we present chromosome-level haploid maps for two A.annua strains with different artemisinin contents to explore the relationships between genomic organization and artemisinin production.High-fidelity sequencing,optical mapping,and chromatin conformation capture sequencing were used to assemble the heterogeneous and repetitive genome and resolve the haplotypes of A.annua.Approximately 5o,ooo genes were annotated for each haplotype genome,and a triplication event that occurred approximately 58.12million years ago was examined for the first time in this species.A total of 3,903,467-5,193,414 variants(SNPs,indels,and structural variants)were identified in the 1.5-Gb genome during pairwise comparison between haplotypes,consistent with the high heterozygosity of this species.Genomic analyses revealed a correlation between artemisinin concents and the copy number of amorpha-4,11-dienes ynthasegenes.This correlation was further confirmed by resequencing of 36A.annua samples with varied artemisinin contents.Circular consensus sequencing of transcripts facilitated the detection of paralog expression.Collectively,our study provides chromosome-level allele-aware genome assemblies for two A.annua strains and new insights into the biosynthesis of artemisinin and its regulation,which will contribute to conquering malaria worldwide.