Biosynthesis Of Amorpha-4, 11-diene, A Precursor Of The Antimalarial Agent Artemisinin, In Escherichia Coli Through Introducing MVA Pathway

Artemisinin-based combination therapies (ACTs) were recommended to be the most effective therapies for the first-line treatment of uncomplicated falciparum malaria. However, artemisinin is often in short supply and unaffordable to most malaria patients that limits the wide use of ACTs. In this work, production of amorpha-4,11-diene, an artemisinin precursor, was investigated by engineering a heterologous isoprenoid biosynthetic pathway in Escherichia coli. The production of amorpha-4,11-diene was achieved by expression of a synthetic amorpha-4,11-diene synthase gene in E. coli DHGT7 and further improved by about 13.3 fold through introducing the mevalonate pathway from Enterococcus faecalis. After eliminating three pathway bottlenecks including amorpha-4,11-diene synthase, HMG-CoA reducase and mevalonate kinase by optimizing the metabolic flux, the yield of amorpha-4,11-diene was increased by nearly 7.2 fold and reached at 235 mg/L in shaking flask culture. In conclusion, an engineered E. coli was constructed for high-level production of amorpha-4,11-diene.In addition, the establishment of the thin layer chromatography (TLC) for rapid semi-quantitative detection of the production of mevalonate, and the gas chromatography and mass spectrometry linked (GC-MS) for quantitative analysis of the production of mevalonate and amorpha-4,11-diene, provided technical supports for the production of metabolites and regulation of metabolic pathway.