Malaria is still a global concern with around 214 million annual cases and 430,000 annual deaths, mainly among of children younger than 5 (World Health Organization [WHO], 2016). This fatal disease is caused by Plasmodium sp. particularly Plasmodium falciparum that proliferate in female Anopheles mosquitoes (Cox, 2010). Since the 1940s there has been continuous attempts to halt the spread of the disease and this has succeeded in Europe, North America, and parts of Asia and Latin America (Carter and Mendis, 2002). However, not in Sub-Saharan Africa where 80% of the annual malaria patients are found. Besides measures such as vector control and insecticide-treated nets, research and development has led to new drugs and a vaccine. The current preferred therapy is artemisinin combination therapy (ACT) (Banek et al., 2014; Lalloo et al., 2016) that is based on artemisinin produced in the natural source Artemisia annua. Artemisinin can also be produced heterologously in the plants Nicotiana benthamiana and Physcomitrella patens (Han et al., 2016; Wang et al., 2016; Ikram et al., 2017). The vaccine toward Plasmodium is called PfSPZ and can be produced in N. benthamiana and P. patens plants (Rosales-Mendoza et al., 2014, 2017; Boes et al., 2016; Epstein et al., 2017).

Malaria drugs have contributed significantly to the reductions in malaria mortality and morbidity. The focus for many years has been to screen traditional medicine to find new antimalarial drugs (Simonsen et al., 2001; Adia et al., 2016; Nondo et al., 2017). The malaria drug artemisinin is an example of this and originates from A. annua, a Chinese medicinal plant (Qinghao), commonly known as sweet wormwood. It was discovered by the Chinese researcher You-You Tu and her team in 1972, and was named Qinghaosu (Klayman, 1985; Tu, 2011). Chemically, artemisinin is a sesquiterpene lactone with a unique endoperoxide structure, without the nitrogen containing heterocyclic ring like other antimalarial compounds (Luo and Shen, 1987). The in planta accumulation of artemisinin is 0.01–1.4% dry weight depending on the plant variety and artemisinin is stored in the glandular trichomes of A. annua (Duke et al., 1994; Van Agtmael et al., 1999; Bhakuni et al., 2001; Muangphrom et al., 2016). The current production using plants with a “low” content of artemisinin can only just cover the global need, which have led to an increase in price (Peplow, 2016). In 2006, World Health Organization (WHO) recommended artemisinin as the first-choice treatment for malaria. Rapid emergence of antimalarial drug resistance drew attention to formulation of artemisinin-based combination therapy (ACT) with artemisinin as the primary substance and is now the preferred treatment (World Health Organization [WHO], 2015).

To secure the global need of artemisinin, there are continuous and extensive efforts to enhance the production of artemisinin in the native plant A. annua. A. annua is currently the primary commercial source of artemisinin and significant breeding programs has contributed to higher artemisinin content in the plant (Ma et al., 2015; Pulice et al., 2016; Xie et al., 2016), including establishment of mutant libraries (Pandey et al., 2016). Several plant-breeding techniques have been applied to create superior cultivars of A. annua. For example, conventional breeding by crossing A. annua with high artemisinin content in wild population has led to hybrid lines with 2% artemisinin d.w (Delabays et al., 2001; Cockram et al., 2012). A detailed genetic map of A. annua comprising of genes and markers controlling artemisinin yield has been established to generate robust high yielding crops (Graham et al., 2010). Identification of A. annua superior parental lines with desired traits from these genetic maps has provided two high-yielding hybrids and diallel crossing of the parental lines and the hybrids has showed consistent results for the development of improved A. annua hybrids (Townsend et al., 2013). Doubling the number of chromosomes generated a new variety of tetraploid cultivar with higher artemisinin content and this might become a new elite line (Banyai et al., 2010b). The overall production of the new cultivars from various laboratories have increased the level of artemisinin to about 1 to 2% d.w. (Delabays et al., 1993; Ferreira et al., 2005; Graham et al., 2010; Brisibe et al., 2012), but not all the established plant lines are stable over generations (Delabays et al., 2001).

Efforts in plant breeding have been challenging due to the heterozygous nature of A. annua, which results in transgenic plants with varying degrees of artemisinin content even though they were generated in the same laboratory (Delabays et al., 2001; Graham et al., 2010; Larson et al., 2013). This variation is due to the segregation of the heterozygous wild type progeny leading to a different genetic background than the parent plant. Although several high content lines have been created, the unstable yield in the progeny of these cultivars were insufficient to increase the global supply of artemisinin (Shretta and Yadav, 2012; Paddon et al., 2013).

Accumulation of artemisinin in A. annua is limited to the small 10 cell glandular trichomes (GT) mostly on leaves and other aerial parts (Ferreira and Janick, 1995; Lommen et al., 2006; Ling et al., 2016). Low GT numbers are correlated to low artemisinin content (Graham et al., 2010; Kjær et al., 2012). Attempts to increase the number of GTs by physical and chemical stress have not been successful (Kjær et al., 2012). One study expressed the β glucosidase (bgl1) gene in A. annua through Agrobacterium-mediated transformation, which resulted in an increase of GT density by 20% on leaves and 66% on flowers and an increase in artemisinin content of 1.4% in leaves and 2.56% in flowers (d.w). Manipulating GT density together with biosynthetic pathway engineering may further increase artemisinin content in A. annua. In depth understanding of A. annua GT generation at the molecular level, will broaden the opportunities of increasing the artemisinin production. This approximately though require a greater acceptance of GMO crops in open fields to ensure the global supply.

Plant tissue culture has also been investigated to establish a production of artemisinin in A. annua hairy root or cell suspension cultures (Nair et al., 1986; Baldi and Dixit, 2008). Several manipulations of the growth conditions such as different sugar supply, light irradiation, UV-B radiation and chilling treatment have led to production of artemisinin in A. annua tissue cultures (Woerdenbag et al., 1993; Liu et al., 2002; Wang and Weathers, 2007; Baldi and Dixit, 2008; Yin et al., 2008; Pandey and Pandey-Rai, 2014). Generating somaclonal variants tolerant against salt stress through gamma-rays irradiation has resulted in 13 somaclonal variants (ASV1 to ASV13) of which one of the variants, ASV12 is a stable salt-tolerant line with a higher expression profile of artemisinin key genes (ADS, CYP71AV1, DBR2, and ALDH1) and a higher artemisinin content as compared to wild type. In addition, treatments with elicitors such as methyl jasmonate has significantly increased artemisinin production by up to 49% including up-regulating the expression of artemisinin biosynthesis genes as well as increased GT index (0.128) (Baldi and Dixit, 2008; Wang et al., 2010; Dangash et al., 2014; Xiang et al., 2015). Other elicitors such as chitosan, gibberellic acid, and salicylic acid also aid in the accumulation of artemisinin (Guo et al., 2010; Banyai et al., 2011). Combinations of various cultivation and elicitation methods are currently being geared for a mass production of artemisinin in A. annua hairy roots via bioreactors with 6.3 g/L dry weight (37.50 g fresh weight) biomass and 0.32 mg/g artemisinin content after 25 days (Patra and Srivastava, 2017).

Other efforts to enhance artemisinin production have been attempted through genetic engineering of the artemisinin biosynthetic pathway genes in microbial heterologous hosts. Extensive work on the development of microbial production of artemisinin precursors led to semi-synthesis of artemisinin, but this is only partly commercially successful (Benjamin et al., 2016; Peplow, 2016; Singh et al., 2017). In this review, the progress and recent bioengineering advances in artemisinin production in stable heterologous in planta systems including genetic modifications of A. annua is summarized.

The biosynthesis of Artemisinin (Figure 1) has been explored for many years. However, not every detail about the regulation and biosynthesis is completely understood, but the discovery that the whole biosynthesis is located in the glandular trichomes of A. annua has facilitated in-depth regulatory studies (Olsson et al., 2009; Olofsson et al., 2011). Derived from the general terpenoid biosynthesis, two molecules of isopentenyl diphosphate (IPP) and one dimethylallyl diphosphate (DMAPP) are condensed by farnesyl diphosphate synthase (FPPS/FPS) into farnesyl diphosphate (FPP, farnesyl pyrophosphate), the C15 sesquiterpenoid precursor (Weathers et al., 2006; Brown, 2010; Wen and Yu, 2011). Overexpression of FPS in A. annua resulted in an increase of artemisinin production (Han et al., 2006; Banyai et al., 2010a), which confirms the role of FPS and availability of the substrates in the regulation of artemisinin biosynthesis similar to other sesquiterpene lactones (Simonsen et al., 2013).

FPP is converted to amorpha-4,11-diene by amorpha-4,11-diene synthase (ADS) via carbocation formation and cyclization (Bouwmeester et al., 1999; Mercke et al., 2000; Picaud et al., 2005, 2006). In the following two oxidization steps, amorpha-4,11-diene is hydroxylated into artemisinic alcohol and oxidized to artemisinic aldehyde by amorphadiene monooxygenase (CYP71AV1), a cytochrome P450 enzyme (Teoh et al., 2006; Wang et al., 2011). The activity of the CYP71AV1 has also been confirmed through a knock-out of the endogenous gene in A. annua showing that these plants do not produce any downstream products of amorphadiene (Czechowski et al., 2016). It has later been discovered that the alcohol dehydrogenase (ADH1, a dehydrogenase/reductase enzyme) is specific toward artemisinin alcohol and oxidizes this to the aldehyde. This specificity and strong expression in A. annua glandular trichomes confirms that ADH1 is responsible for oxidation of artemisinic alcohol to artemisinic aldehyde (Olofsson et al., 2011; Paddon et al., 2013; He et al., 2017). Artemisinic aldehyde is further reduced to dihydroartemisinic aldehyde by artemisinic aldehyde Δ11 (13) reductase (DBR2) and subsequently oxidized to dihydroartemisinic acid by aldehyde dehydrogenase (ALDH1), which is also expressed in the trichomes (Zhang et al., 2008; Teoh et al., 2009; Rydén et al., 2010; Liu et al., 2016). Besides catalyzing the oxidation of dihydroartemisinic aldehyde to the acid, ALDH1 also catalyzes the oxidation of artemisinic aldehyde to artemisinic acid (a reaction that in yeast is catalyzed by CYP71AV1) (Teoh et al., 2006, 2009). Another enzyme, dihydroartemisinic aldehyde reductase (RED1) converts dihydroartemisinic aldehyde to dihydroartemisinic alcohol, a “dead end” substance, which affects the production yield of artemisinin (Rydén et al., 2010). The final step is a light-induced non-enzymatic spontaneous reaction converting dihydroartemisinic acid to artemisinin and artemisinic acid to arteannuin B (Sy and Brown, 2002; Teoh et al., 2006; Czechowski et al., 2016).

Current advances in genetic and metabolic engineering drive a more diverse research and development approach on improving in planta production of artemisinin. The successes achieved in heterologous plant hosts and engineering of A. annua remains are of great importance. Microbial engineering of artemisinic acid shows some potential, but the added costs for later chemical synthesis of artemisinin is a detracting factor for replacing A. Annua as the main artemisinin source. Progress in plant engineering and synthetic biology has significantly improved the awareness of using plant as production hosts leading to great efforts in the implementation and enhancement of artemisinin production in both in vivo and in vitro production. Furthermore, heterologous in planta production seems to be more cost effective and environmentally friendly than other current biotechnological platforms. Advances in multigene transformation, transcription factors along with targeting of cellular compartment techniques will enable elevation of production levels in future engineered plants bringing us closer to industrial scale plant factories for artemisinin production. Perhaps the continuous production of artemisinin and other valuable plant metabolites in suspended bioreactor cultures with in situ extraction to avoid cell toxicity is not too far in the future. This will avoid the regulatory restrictions on in field GMO plants, and allow for stable continues production of drugs.

NI and HS collectively wrote the manuscript and initiated the work behind it. NI contributed with major parts of the literature research.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.