Experimental design-aided systematic pathway optimization of glucose uptake and deoxyxylulose phosphate pathway for improved amorphadiene production
Artemisinin is a potent antimalarial drug; however, it suffers from unstable and insufficient supply from plant source. Here, we established a novel multivariate-modular approach based on experimental design for systematic pathway optimization that succeeded in improving the production of amorphadiene (AD), the precursor of artemisinin, in Escherichia coli. It was initially found that the AD production was limited by the imbalance of glyceraldehyde 3-phosphate (GAP) and pyruvate (PYR), the two precursors of the 1-deoxy-d-xylulose-5-phosphate (DXP) pathway. Furthermore, it was identified that GAP and PYR could be balanced by replacing the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) with the ATP-dependent galactose permease and glucose kinase system (GGS) and this resulted in fivefold increase in AD titer (11 to 60 mg/L). Subsequently, the experimental design-aided systematic pathway optimization (EDASPO) method was applied to systematically optimize the transcriptional expressions of eight critical genes in the glucose uptake and the DXP and AD synthesis pathways. These genes were classified into four modules and simultaneously controlled by T7 promoter or its variants. A regression model was generated using the four-module experimental data and predicted the optimal expression ratios among these modules, resulting in another threefold increase in AD titer (60 to 201 mg/L). This EDASPO method may be useful for the optimization of other pathways and products beyond the scope of this study.
KeywordsAmorphadiene Experimental design-aided systematic pathway optimization Multivariate-modular approach Deoxyxylulose phosphate pathway The phosphotransferase system
We would like to acknowledge the financial support from Singapore-MIT Alliance.
- De Anda R, Lara AR, Hernandez V, Hernandez-Montalvo V, Gosset G, Bolivar F, Ramirez OT (2006) Replacement of the glucose phosphotransferase transport system by galactose permease reduces acetate accumulation and improves process performance of Escherichia coli for recombinant protein production without impairment of growth rate. Metab Eng 8(3):281–90CrossRefPubMedGoogle Scholar
- Flores N, Leal L, Sigala JC, de Anda R, Escalante A, Martinez A, Ramirez OT, Gosset G, Bolivar F (2007) Growth recovery on glucose under aerobic conditions of an Escherichia coli strain carrying a phosphoenolpyruvate:carbohydrate phosphotransferase system deletion by inactivating arcA and overexpressing the genes coding for glucokinase and galactose permease. J Mol Microbiol Biotechnol 13(1–3):105–16CrossRefPubMedGoogle Scholar
- Martínez K, de Anda R, Hernández G, Escalante A, Gosset G, Ramírez OT, Bolívar FG (2008) Coutilization of glucose and glycerol enhances the production of aromatic compounds in an Escherichia coli strain lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system. Microb Cell Fact 7(1):1CrossRefPubMedCentralPubMedGoogle Scholar
- Morrone D, Lowry L, Determan MK, Hershey DM, Xu M, Peters RJ (2010) Increasing diterpene yield with a modular metabolic engineering system in E. coli: comparison of MEV and MEP isoprenoid precursor pathway engineering. Appl Microbiol Biotechnol 85(6):1893–906CrossRefPubMedCentralPubMedGoogle Scholar
- Too HP, ZOU R, Stephanopoulos GN (2014) Univariant extrinsic initiator control system for microbes and an in vitro assembly of large recombinant dna molecules from multiple components. USA Patent WO2014077782 A1, Nov 15, 2013Google Scholar
- Westfall PJ, Pitera DJ, Lenihan JR, Eng D, Woolard FX, Regentin R, Horning T, Tsuruta H, Melis DJ, Owens A, Fickes S, Diola D, Benjamin KR, Keasling JD, Leavell MD, McPhee DJ, Renninger NS, Newman JD, Paddon CJ (2012) Production of amorphadiene in yeast, and its conversion to dihydroartemisinic acid, precursor to the antimalarial agent artemisinin. Proc Natl Acad Sci U S A 109(3):E111–8CrossRefPubMedCentralPubMedGoogle Scholar