Heterologous Production of Artemisinin in Physcomitrium patens by Direct in vivo Assembly of Multiple DNA Fragments.

The sesquiterpene lactone compound artemisinin is a natural medicinal product of commercial importance. This Artemisia annua-derived secondary metabolite is well known for its antimalarial activity and has been studied in several other biological assays. However, the major shortcoming in its production and commercialization is its low accumulation in the native plant. Moreover, the chemical synthesis of artemisinin is difficult and expensive due to its complex structure. Hence, an alternative and sustainable production system of artemisinin in a heterologous host is required. Previously, heterologous production of artemisinin was achieved by Agrobacterium-mediated transformation. However, this requires extensive bioengineering of modified Nicotiana plants. Recently, a technique involving direct in vivo assembly of multiple DNA fragments in the moss, P. patens, has been successfully established. We utilized this technique to engineer artemisinin biosynthetic pathway genes into the moss, and artemisinin was obtained without further modifications with high initial production. Here, we provide protocols for establishing moss culture accumulating artemisinin, including culture preparation, transformation method, and compound detection via HS-SPME, UPLC-MRM-MS, and LC-QTOF-MS. The bioengineering of moss opens up a more sustainable, cost effective, and scalable platform not only in artemisinin production but also other high-value specialized metabolites in the future.

Stability Analysis of the Asiatic Acid-COX-2 Complex Using 100 ns Molecular Dynamic Simulations and Its Selectivity against COX-2 as a Potential Anti-Inflammatory Candidate.

Asiatic acid, a triterpenoid compound, has been shown to have anti-inflammatory activity through the inhibition of the formation of cyclooxygenase-2 (COX-2) in vitro and in vivo. This study was conducted to determine the binding stability and the inhibitory potential of asiatic acid as an anti-inflammatory candidate. The study involved in vitro testing utilizing a colorimetric kit as well as in silico testing for the pharmacophore modeling and molecular dynamic (MD) simulation of asiatic acid against COX-2 (PDB ID: 3NT1). The MD simulations showed a stable binding of asiatic acid to COX-2 and an RMSD range of 1-1.5 Å with fluctuations at the residues of Phe41, Leu42, Ile45, Arg44, Asp367, Val550, Glu366, His246, and Gly227. The total binding energy of the asiatic acid-COX-2 complex is -7.371 kcal/mol. The anti-inflammatory activity of the asiatic acid inhibition of COX-2 was detected at IC50 values of 120.17 µM. Based on pharmacophore modeling, we discovered that carboxylate and hydroxyl are the two main functional groups that act as hydrogen bond donors and acceptors interacting with the COX-2 enzyme. From the results, it is evident that asiatic acid is a potential anti-inflammatory candidate with high inhibitory activity in relation to the COX-2 enzyme.

Insights into Heterologous Biosynthesis of Arteannuin B and Artemisinin in Physcomitrella patens.

: 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.

A virtual screening approach for identifying plants with anti H5N1 neuraminidase activity.

Recent outbreaks of highly pathogenic and occasional drug-resistant influenza strains have highlighted the need to develop novel anti-influenza therapeutics. Here, we report computational and experimental efforts to identify influenza neuraminidase inhibitors from among the 3000 natural compounds in the Malaysian-Plants Natural-Product (NADI) database. These 3000 compounds were first docked into the neuraminidase active site. The five plants with the largest number of top predicted ligands were selected for experimental evaluation. Twelve specific compounds isolated from these five plants were shown to inhibit neuraminidase, including two compounds with IC50 values less than 92 µM. Furthermore, four of the 12 isolated compounds had also been identified in the top 100 compounds from the virtual screen. Together, these results suggest an effective new approach for identifying bioactive plant species that will further the identification of new pharmacologically active compounds from diverse natural-product resources.