Applied Microbiology and Biotechnology

, Volume 99, Issue 9, pp 3825–3837 | Cite as

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

  • Congqiang Zhang
  • Ruiyang Zou
  • Xixian Chen
  • Gregory Stephanopoulos
  • Heng-Phon Too
Biotechnological products and process engineering


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.


Amorphadiene 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.

Supplementary material

253_2015_6463_MOESM1_ESM.pdf (402 kb)
ESM 1 (PDF 401 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Congqiang Zhang
    • 1
    • 2
  • Ruiyang Zou
    • 1
  • Xixian Chen
    • 1
    • 2
  • Gregory Stephanopoulos
    • 1
    • 3
  • Heng-Phon Too
    • 1
    • 2
  1. 1.Chemical and Pharmaceutical EngineeringSingapore-MIT AllianceSingaporeSingapore
  2. 2.Department of Biochemistry, Yong Loo Lin School of MedicineNational University of SingaporeSingaporeSingapore
  3. 3.Department of Chemical EngineeringMassachusetts Institute of TechnologyCambridgeUSA

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