High-Level Production of Amorpha-4,11-Diene, a Precursor of the Antimalarial Agent Artemisinin, in Escherichia coli

Abstract

BackgroundArtemisinin derivatives are the key active ingredients in Artemisinin combination therapies (ACTs), the most effective therapies available for treatment of malaria. Because the raw material is extracted from plants with long growing seasons, artemisinin is often in short supply, and fermentation would be an attractive alternative production method to supplement the plant source. Previous work showed that high levels of amorpha-4,11-diene, an artemisinin precursor, can be made in Escherichia coli using a heterologous mevalonate pathway derived from yeast (Saccharomyces cerevisiae), though the reconstructed mevalonate pathway was limited at a particular enzymatic step.Methodology/ Principal FindingsBy combining improvements in the heterologous mevalonate pathway with a superior fermentation process, commercially relevant titers were achieved in fed-batch fermentations. Yeast genes for HMG-CoA synthase and HMG-CoA reductase (the second and third enzymes in the pathway) were replaced with equivalent genes from Staphylococcus aureus, more than doubling production. Amorpha-4,11-diene titers were further increased by optimizing nitrogen delivery in the fermentation process. Successful cultivation of the improved strain under carbon and nitrogen restriction consistently yielded 90 g/L dry cell weight and an average titer of 27.4 g/L amorpha-4,11-diene.Conclusions/ SignificanceProduction of >25 g/L amorpha-4,11-diene by fermentation followed by chemical conversion to artemisinin may allow for development of a process to provide an alternative source of artemisinin to be incorporated into ACTs.

A squalene synthase protein degradation method for improved sesquiterpene production in Saccharomyces cerevisiae

“…To optimize heterologous sesquiterpene production in yeast, it is necessary to increase metabolic flux flowing through the MVA pathway, and to redirect flux from competing native isoprenoid production towards the desired end product (Asadollahi et al, 2008;Paddon et al, 2013;Tsuruta et al, 2009;Vickers et al, 2015a;Yoon et al, 2009). In metabolic engineering, transcriptional regulation is commonly employed to control enzyme levels (and consequently, metabolic flux) in desired pathways.…”

“…In order to increase availability of FPP in the presence of a heterologous sesquiterpene synthase, the entire MVA pathway as well as FPP synthase are typically overexpressed. This strategy is used both in yeast and in E. coli, the latter of which uses the alternative methylerythritol phosphate pathway for native isoprenoid production (Bongers et al, 2015;Ignea et al, 2011;Tsuruta et al, 2009;Yoon et al, 2009). In addition to the low capacity for prenyl phosphate (IPP/DMAPP/FPP) synthesis, non-productive consumption of FPP for sterol synthesis is a limitation for heterogeneous sesquiterpene production.…”

Experimental design-aided systematic pathway optimization of glucose uptake and deoxyxylulose phosphate pathway for improved amorphadiene production

“…AD quantification AD was extracted by diluting 5 μL dodecane phase into 495 μL ethyl acetate and analyzed on an Agilent 7980A gas chromatograph equipped with an Agilent 5975C mass spectrometer (GC/MS). Beta-caryophyllene (Sigma-Aldrich) was used as an equivalent, and the GC-MS condition was set as the method reported by scanning for only two ions (204 and 189 m/z ion) (Tsuruta et al 2009). …”

A platform pathway for production of 3-hydroxyacids provides a biosynthetic route to 3-hydroxy-γ-butyrolactone

“…Khosla and colleagues 50 reported titres of B25 mg l À 1 in the initial reconstruction of polyketide synthesis in E.coli, while the Keasling group obtained maximum amorphadiene titres of 112 mg l À 1 in the first reported synthesis in an engineered organism 51 . These low titres were observed in spite of the availability of natural pathways to produce the target molecules.…”