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The plant Artemisia annua L. is famous for producing "artemisinin", which is an essential component in the treatment of malaria. The glandular secretory trichomes (GSTs) on the leaves of A. annua secrete and store artemisinin. Previous research has demonstrated that raising GST density can effectively raise artemisinin content. However, the molecular mechanism of GST initiation is not fully understood yet. In this study, we identified an MYB transcription factor, the AaMYB108-like , which is co-induced by light and jasmonic acid, and positively regulates glandular secretory trichome initiation in A. annua . Overexpression of the AaMYB108-like gene in A. annua increased GST density and enhanced the artemisinin content, whereas anti-sense of the AaMYB108-like gene resulted in the reduction in GST density and artemisinin content. Further experiments demonstrated that the AaMYB108-like gene could form a complex with AaHD8 to promote the expression of downstream AaHD1 , resulting in the initiation of GST. Taken together, the AaMYB108-like gene is a positive regulator induced by light and jasmonic acid for GST initiation in A. annua.

Trichomes are attractive cells for terpenoid biosynthesis and accumulation in Artemisia annua . However, the molecular process underlying the trichome of A. annua is not yet fully elucidated. In this study, an analysis of multi-tissue transcriptome data was performed to examine trichome-specific expression patterns. A total of 6646 genes were screened and highly expressed in trichomes, including artemisinin biosynthetic genes such as amorpha-4,11-diene synthase ( ADS ) and cytochrome P450 monooxygenase ( CYP71AV1 ). Mapman and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that trichome-specific genes were mainly enriched in lipid metabolism and terpenoid metabolism. These trichome-specific genes were analyzed by a weighted gene co-expression network analysis (WGCNA), and the blue module linked to terpenoid backbone biosynthesis was determined. Hub genes correlated with the artemisinin biosynthetic genes were selected based on TOM value. ORA , Benzoate carboxyl methyltransferase ( BAMT ), Lysine histidine transporter-like 8 ( AATL1 ), Ubiquitin-like protease 1 ( Ulp1 ) and TUBBY were revealed as key hub genes induced by methyl jasmonate (MeJA) for regulating artemisinin biosynthesis. In summary, the identified trichome-specific genes, modules, pathways and hub genes provide clues and shed light on the potential regulatory mechanisms of artemisinin biosynthesis in trichomes in A. annua .

Malaria is a life-threatening disease. Artemisinin-based combination therapy (ACT) is the preferred choice for malaria treatment recommended by the World Health Organization. At present, the main source of artemisinin is extracted from Artemisia annua ; however, the artemisinin content in A. annua is only 0.1-1%, which cannot meet global demand. Meanwhile, the chemical synthesis of artemisinin has disadvantages such as complicated steps, high cost and low yield. Therefore, the application of the synthetic biology approach to produce artemisinin in vivo has magnificent prospects. In this review, the biosynthesis pathway of artemisinin was summarized. Then we discussed the advances in the heterologous biosynthesis of artemisinin using microorganisms ( Escherichia coli and Saccharomyces cerevisiae ) as chassis cells. With yeast as the cell factory, the production of artemisinin was transferred from plant to yeast. Through the optimization of the fermentation process, the yield of artemisinic acid reached 25 g/L, thereby producing the semi-synthesis of artemisinin. Moreover, we reviewed the genetic engineering in A. annua to improve the artemisinin content, which included overexpressing artemisinin biosynthesis pathway genes, blocking key genes in competitive pathways, and regulating the expression of transcription factors related to artemisinin biosynthesis. Finally, the research progress of artemisinin production in other plants ( Nicotiana , Physcomitrella , etc.) was discussed. The current advances in artemisinin biosynthesis may help lay the foundation for the remarkable up-regulation of artemisinin production in A. annua through gene editing or molecular design breeding in the future.

Artemisinin is an anti-malarial sesquiterpene lactone derived from Artemisia annua L. (Asteraceae family). One of the most widely used modes of treatment for malaria is an artemisinin-based combination therapy. Artemisinin and its associated compounds have a variety of pharmacological qualities that have helped achieve economic prominence in recent years. So far, research on the biosynthesis of this bioactive metabolite has revealed that it is produced in glandular trichomes and that the genes responsible for its production must be overexpressed in order to meet demand. Using biotechnological applications such as tissue culture, genetic engineering, and bioreactor-based approaches would aid in the upregulation of artemisinin yield, which is needed for the future. The current review focuses on the tissue culture aspects of propagation of A. annua and production of artemisinin from A. annua L. cell and organ cultures. The review also focuses on elicitation strategies in cell and organ cultures, as well as artemisinin biosynthesis and metabolic engineering of biosynthetic genes in Artemisia and plant model systems.

Artemisia annua is an important medicinal plant producing the majority of the antimalarial compound artemisinin. Jasmonates are potent inducers of artemisinin accumulation in Artemisisa annua plants. As the receptor of jasmonates, the F-box protein COI1 is critical to the JA signaling required for plant development, defense, and metabolic homeostasis. AaCOI1 from Artemisia annua, homologous to Arabidopsis AtCOI1, encodes a F-box protein located in the nuclei. Expressional profiles of the AaCOI1 in the root, stem, leaves, and inflorescence was investigated. The mRNA abundance of AaCOI1 was the highest in inflorescence, followed by in the leaves. Upon mechanical wounding or MeJA treatment, expression of AaCOI1 was upregulated after 6 h. When ectopically expressed, driven by the native promoter from Arabidopsis thaliana, AaCOI1 could partially complement the JA sensitivity and defense responses, but fully complemented the fertility, and the JA-induced anthocyanin accumulation in a coi1-16 loss-of-function mutant. Our study identifies the paralog of AtCOI1 in Artemisia annua, and revealed its implications in development, hormone signaling, defense, and metabolism. The results provide insight into JA perception in Artemisia annua, and pave the way for novel molecular breeding strategies in the canonical herbs to manipulate the anabolism of pharmaceutic compounds on the phytohormonal level., Competing Interests: The authors declare no conflicts of interest.

: Metabolic engineering is an integrated bioengineering approach, which has made considerable progress in producing terpenoids in plants and fermentable hosts. Here, the full biosynthetic pathway of artemisinin, originating from Artemisia annua , was integrated into the moss Physcomitrella patens . Different combinations of the five artemisinin biosynthesis genes were ectopically expressed in P. patens to study biosynthesis pathway activity, but also to ensure survival of successful transformants. Transformation of the first pathway gene, ADS , into P. patens resulted in the accumulation of the expected metabolite, amorpha-4,11-diene, and also accumulation of a second product, arteannuin B. This demonstrates the presence of endogenous promiscuous enzyme activity, possibly cytochrome P450s, in P. patens . Introduction of three pathway genes, ADS-CYP71AV1-ADH1 or ADS-DBR2-ALDH1 both led to the accumulation of artemisinin, hinting at the presence of one or more endogenous enzymes in P. patens that can complement the partial pathways to full pathway activity. Transgenic P. patens lines containing the different gene combinations produce artemisinin in varying amounts. The pathway gene expression in the transgenic moss lines correlates well with the chemical profile of pathway products. Moreover, expression of the pathway genes resulted in lipid body formation in all transgenic moss lines, suggesting that these may have a function in sequestration of heterologous metabolites. This work thus provides novel insights into the metabolic response of P. patens and its complementation potential for A. annua artemisinin pathway genes. Identification of the related endogenous P. patens genes could contribute to a further successful metabolic engineering of artemisinin biosynthesis, as well as bioengineering of other high-value terpenoids in P. patens ., Competing Interests: All authors declare that they have no conflict of interest. HTS is co-founder of Mosspiration Biotech IVS that aim to produce fragrances in P. patens, but not artemisinin.

Artemisinin, which has been isolated from Artemisia annua L., is the most effective antimalarial drug and has saved millions of lives. In addition, artemisinin and its derivatives have anti-tumor, anti-parasitic, anti-fibrosis, and anti-arrhythmic properties, which enhances the demand for these compounds. Improving the content of artemisinin in A. annua is therefore becoming an increasing research interest, as the chemical synthesis of this metabolite is not viable. Ultraviolet B and C irradiation have been reported to improve the artemisinin content in A. annua , but they are harmful to plant growth and development. Therefore, we screened other light sources to examine if they could promote artemisinin content without affecting plant growth and development. We found that red and blue light could enhance artemisinin accumulation by promoting the expression of the genes that were involved in artemisinin biosynthesis, such as amorpha-4,11-diene synthase (ADS) and cytochrome P450 monooxygenase (CYP71AV1) genes. Thus, in addition to being the main light sources for photosynthesis, red and blue light play a key role in plant secondary metabolism, and optimizing the combination of these light might allow for the productionof artemisinin-rich A. annua .

The complete chloroplast genome of Artemisia annua (Asteraceae), the primary source of artemisinin, was sequenced and analyzed. The A. annua cp genome is 150,995 bp, and harbors a pair of inverted repeat regions (IRa and IRb), of 24,850 bp each that separate large (LSC, 82,988 bp) and small (SSC, 18,267 bp) single-copy regions. Our annotation revealed that the A. annua cp genome contains 113 genes and 18 duplicated genes. The gene order in the SSC region of A. annua is inverted; this fact is consistent with the sequences of chloroplast genomes from three other Artemisia species. Fifteen (15) forward and seventeen (17) inverted repeats were detected in the genome. The existence of rich SSR loci in the genome suggests opportunities for future population genetics work on this anti-malarial medicinal plant. In A. annua cpDNA, the rps19 gene was found in the LSC region rather than the IR region, and the rps19 pseudogene was absent in the IR region. Sequence divergence analysis of five Asteraceae species indicated that the most highly divergent regions were found in the intergenic spacers, and that the differences between A. annua and A. fukudo were very slight. A phylogenetic analysis revealed a sister relationship between A. annua and A. fukudo . This study identified the unique characteristics of the A. annua cp genome. These results offer valuable information for future research on Artemisia species identification and for the selective breeding of A. annua with high pharmaceutical efficacy., Competing Interests: The authors declare no conflict of interest.

Plant cell cultures as valuable tools for the production of specific metabolites can be greatly improved by the application of elicitors including cyclodextrins (CDs) for enhancing the yields of the desired plant compounds. Here the effects of 2,6-dimethyl-β-cyclodextrins (DIMEB) on the production of carotenoids and quinones from Artemisia annua L. cell suspension cultures were investigated. The addition of 50 mM DIMEB induced an early increase of intracellular carotenoid and quinone contents, which could be observed to a higher extent for lutein (10-fold), Q9 (3-fold) and Q10 (2.5-fold). Real Time PCR analysis revealed that the expression of 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) gene in DIMEB treated cell cultures after three days was 2.5-fold higher than in untreated samples, thus suggesting that the DIMEB induced increase of carotenoids and quinones could be due to the induction of the plastidial isoprenoid biosynthetic route. In addition, the DIMEB treatment induced an enhanced release of carotenoids and quinones into the culture medium of A. annua cell suspension cultures possibly due to the ability of CDs to form inclusion complexes with hydrophobic molecules.

The biosynthesis of the valuable sesquiterpene anti-malarial, artemisinin, is known to respond to exogenous sugar concentrations. Here young Artemisia annua L. seedlings (strain YU) were used to measure the transcripts of six key genes in artemisinin biosynthesis in response to growth on sucrose, glucose, or fructose. The measured genes are: from the cytosolic arm of terpene biosynthesis, 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR), farnesyl disphosphate (FPS); from the plastid arm of terpene biosynthesis, 1-deoxyxylulose-5-phosphate synthase (DXS), 1-deoxyxylulouse 5-phosphate reductoisomerase (DXR); from the dedicated artemisinin pathway amorpha-4,11-diene synthase (ADS), and the P450, CYP71AV1 (CYP). Changes in intracellular concentrations of artemisinin (AN) and its precursors, dihydroartemisinic acid (DHAA), artemisinic acid (AA), and arteannuin B (AB) were also measured in response to these three sugars. FPS, DXS, DXR, ADS and CYP transcript levels increased after growth in glucose, but not fructose. However, the kinetics of these transcripts over 14 days was very different. AN levels were significantly increased in glucose-fed seedlings, while levels in fructose-fed seedlings were inhibited; in both conditions this response was only observed for 2 days after which AN was undetectable until day 14. In contrast to AN, on day 1 AB levels doubled in seedlings grown in fructose compared to those grown in glucose. Results showed that transcript level was often negatively correlated with the observed metabolite concentrations. When seedlings were gown in increasing levels of AN, some evidence of a feedback mechanism emerged, but mainly in the inhibition of AA production. Together these results show the complex interplay of exogenous sugars on the biosynthesis of artemisinin in young A. annua seedlings.