Abstract
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.
Highlights
Artemisinin is a potent malaria drug that is exclusively produced in the plant Artemisia annua.The limited production of artemisinin in glandular trichomes of leaves and flowers has led to an extensive cultivation of Artemisia plants to meet the needs of the patients
ADS accumulated arteannuin B, which is thought to be derived from artemisinic acid through photo-oxidation. This suggest accumulation of artemisinic acid in the transgenic lines and reveals a promiscuous activity of an endogenous oxidative enzyme such as the cytochrome P450 in P. patens, which can catalyse the triple oxidation of amorpha-4,11-diene via artemisinic alcohol and aldehyde to the acid
We show that the anti-malaria drug, artemisinin, can be produced in P. patens with either complete or partial introduction and expression of the artemisinin pathway genes
Summary
Artemisinin is a potent malaria drug that is exclusively produced in the plant Artemisia annua. The limited production of artemisinin in glandular trichomes of leaves and flowers has led to an extensive cultivation of Artemisia plants to meet the needs of the patients. Various efforts have been performed to improve the production of artemisinin in the plant. All genes responsible for the biosynthesis of the direct precursor of artemisinin, dihydroartemisinic acid, have been characterized (Scheme 1) [1]. The final conversion of dihydroartemisinic acid to artemisinin is thought to be a light-induced non-enzymatic spontaneous reaction [2]. CYP71AV1 is an important cytochrome P450 enzyme [7] in artemisinin biosynthesis as it catalyses three subsequent oxidations of amorpha-4,11-diene to artemisinic acid, via artemisinic alcohol and artemisinic aldehyde [8].
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