Stable Production of the Antimalarial Drug Artemisinin in the Moss Physcomitrella patens

Abstract

Malaria is a real and constant danger to nearly half of the world’s population of 7.4 billion people. In 2015, 212 million cases were reported along with 429,000 estimated deaths. The World Health Organization recommends artemisinin-based combinatorial therapies, and the artemisinin for this purpose is mainly isolated from the plant Artemisia annua. However, the plant supply of artemisinin is irregular, leading to fluctuation in prices. Here, we report the development of a simple, sustainable, and scalable production platform of artemisinin. The five genes involved in artemisinin biosynthesis were engineered into the moss Physcomitrella patens via direct in vivo assembly of multiple DNA fragments. In vivo biosynthesis of artemisinin was obtained without further modifications. A high initial production of 0.21 mg/g dry weight artemisinin was observed after only 3 days of cultivation. Our study shows that P. patens can be a sustainable and efficient production platform of artemisinin that without further modifications allow for industrial-scale production. A stable supply of artemisinin will lower the price of artemisinin-based treatments, hence become more affordable to the lower income communities most affected by malaria; an important step toward containment of this deadly disease threatening millions every year.

Functional expression and characterization of cinnamic acid 4-hydroxylase from the hornwort Anthoceros agrestis in Physcomitrella patens

“…Physcomitrella patens as a novel expression system has already been used for the heterologous expression of enzymes involved in plant specialized metabolism (Anterola et al 2009;Bach et al 2014;Zhan et al 2014;Pan et al 2015;Khairul Ikram et al 2017). Stable transformants can be obtained due to the high rates of homologous recombination making the permanent use of selective media unnecessary (Schaefer et al 1991).…”

“…Some genes of plant specialized metabolism have already been successfully expressed in Physcomitrella patens, e.g., taxadiene synthase from Taxus brevifolia (Anterola et al 2009), sclareol synthase from Salvia sclarea (Pan et al 2015) and patchoulol synthase from Pogostemon cablin or santalene synthase from Santalum album (Zhan et al 2014). The full biosynthetic system for artemisinin production, including a cytochrome P450-catalyzed step, has also successfully been transferred to Physcomitrella patens (Khairul Ikram et al 2017).…”

Traps and Pitfalls—Unspecific Reactions in Metabolic Engineering of Sesquiterpenoid Pathways

“…This lack of an endogenous glycosyl transferase in this specific subcellular compartment may be the reason why no diglucose-conjugates were formed. Expression of the metabolic pathway to artemisinic acid in Physcomitrella patens resulted in the accumulation of nongylcosylated artemisinic acid in the apoplast [18]. Therefore, if glycosylation to an acid-group appears, a promising strategy would be to target a different subcellular localization or to change the expression system.…”

“…The elucidation and metabolic engineering of their biosynthetic pathways have made significant progress in the past decades, as many sesquiterpenes are of commercial interest as fragrances [1], biodiesel [2] or pharmaceuticals [3,4]. The reconstruction of sesquiterpenoid metabolic pathways has been carried out in various prokaryotic and eukaryotic host models such as Escherichia coli [5,6], Saccharomyces cervisiae [4,[7][8][9][10][11][12], Nicotiana benthamiana [7,8,[13][14][15][16] and Physcomitrella patens [17,18]. The analysis of sesquiterpenes is usually performed by gas or liquid chromatography coupled with mass spectrometry, depending on the volatility of the compound.…”

Engineering modular diterpene biosynthetic pathways in Physcomitrella patens

“…However, each of those platforms required the labor intensive generation of highly specialized lines and lacked the flexibility required for formation of multiple diterpenoid targets. Building on the most recent demonstration of efficient transformation of P. patens and in vivo assembly of multiple linear DNA parts for production of amorphadiene, and Artemisinin (Khairul Ikram et al 2017;King et al 2016), we set out to develop a modular transformation system for diterpenes, imitating their natural formation by pairs of diterpene synthases (diTPSs), and origin of structural diversity through different combinations of diTPSs.…”