Cloning, identification, and in silico analysis of terpene synthases involved in the competing pathways of artemisinin biosynthesis pathway in Artemisia annua L

Abstract

Background: Endoperoxide sesquiterpene lactone, artemisinin, is a widely used antimalarial drug. Artemisia annua L. synthesizes this terpenoid and is the only source of artemisinin. In plants, the content of artemisinin is low (0.1–0.8% by dry weight). One of the best approaches to increase artemisinin production is metabolic engineering. Methods: Both the genes were amplified and cloned in Topo vector. Using computational approach, full gene sequencing and a detailed in silico analysis was performed to check the functional and structural properties of these enzymes. Expression patterns of both the genes were assessed at different developmental stages (vegetative, preflowering, flowering, and postflowering stage) of the plant reverse transcription polymerase chain reaction. Results: Deduced amino acid sequence of these genes possessed two important and highly conserved aspartate-rich motifs, and lacks an N-terminal signal peptide, a characteristic of sesquiterpene synthases. Physiochemical properties demonstrated are thermostable. Low hydropathy values ascertain them to be hydrophobic and are active at neutral pH. Structural analysis disclosed that both the proteins possess more α-helices followed by random coils. Ramachandran analysis showed a C-score of −0.35, TM-score of 0.67 ± 0.13 for β caryophyllene synthase model while as C-score of −0.21, TM-score of 0.69 ± 0.12 for β-Farnesene synthase model. Both the proteins contain enormous nitrosylation sites suggesting their functional link through nitrosylation. Gene expression pattern of both the enzymes were upregulated during preflowering and flowering stage. Conclusion: A thorough analysis of these two putative genes in A. annua L paves way to essential insights concerning terpene biosynthesis in general and regulation in artemisinin production in particular. This study also strongly indicates that these two enzymes are developmentally controlled and may have the regulatory effects on the terpene biosynthesis.